Methods of modulating apoptosis by administration of relaxin agonists or antagonists

ABSTRACT

The present invention relates to the discovery that relaxin is associated with the development or maturation of body tissues. Knockouts of the gene encoding relaxin result in various abnormalities in the development of various tissues. The present invention provides methods of modulating apoptosis by administering a relaxin agonist or antagonist to a subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/945,685, filed Nov. 12, 2010, now U.S. Pat. No. 8,119,136, which is adivisional of U.S. application Ser. No. 11/781,872, filed Jul. 23 2007,now U.S. Pat. No. 7,833,526, which is a divisional of U.S. applicationSer. No. 10/398,553, filed Sep. 22, 2003, now abandoned, which is a U.S.national stage application under 35 U.S.C. §371 of InternationalApplication No. PCT/US01/42484, filed Oct. 4, 2001, which claimspriority from Provisional Application No. 60/238,232 filed Oct. 4, 2000,Provisional Application No. 60/241,991 filed Oct. 20, 2000, andProvisional Application No. 60/242,037, filed Oct. 20, 2000, thedisclosures of which are incorporated herein by reference.

BACKGROUND

The growth and development of normal tissues is achieved by programmedcell proliferation, differentiation and cell death. Cell proliferationand differentiation are required for the formation of new cells andtissues. Conversely, programmed cell death, also referred to asapoptosis, is required to remove existing cells, including immature ordamaged cells. Apoptosis naturally occurs in virtually all tissues ofthe body. Apoptosis plays a critical role in tissue homeostasis, thatis, it ensures that the number of new cells produced are correspondinglyoffset by an equal number of cells that die. For example, the cells inthe intestinal lining divide so rapidly that the body must eliminatecells after only three days in order to prevent the overgrowth of theintestinal lining.

The disruption of the genetic program, by either abnormally increasingor decreasing rate of cell proliferation and/or apoptosis can result inabnormal tissue development. For example, decreases in cellproliferation below normal levels can lead to immature tissues and othertissue abnormalities. Increases in cell proliferation above normallevels are thought to be major events in the development of neoplasiaand cancer, as well as other cell proliferative disorders. Abnormalincreases in apoptosis can also lead to precancerous lesions.Precancerous lesions include lesions of the breast (that can developinto breast cancer), lesions of the prostate (that can develop intoprostate cancer) or skin (that can develop into malignant melanoma orbasal cell carcinoma), colonic adenomatous polyps (that can develop intocolon cancer), and other such neoplasia. Such lesions exhibit a strongtendency to develop into malignant tumors or cancer.

Precancerous lesions can result from an accumulation of insults toexisting cells in various tissues of the body. Such insults can includeexposure to sunlight, radiation, mutagens and carcinogens normally foundin the diet, chemicals such as pesticides, herbicides, preservatives,and the like. These insults can result in the accumulation of mutationsin the cells, which can lead to hyperplastic conditions (i.e., abnormalincreases in cell number), such as, for example, hyperplasia of liver,kidney, spleen, thymus, intestine, lung or prostate tissues. Thedown-regulation of apoptosis can also lead to the accumulation of cellsin these hyperplastic conditions.

An abnormal increase in apoptosis can interfere with normal developmentand/or differentiation of tissues. For example, apoptosis is requiredduring pregnancy and for maturation of the male reproductive tracttissues. An abnormal increase in apoptosis can also interfere with theformation of new cells and tissues, thereby preventing normal tissuematuration or development.

Thus, there is a need for methods of modulating apoptosis byadministering agonists or antagonists of apoptosis. In particular, thereis a need for methods of treating conditions associated, directly orindirectly, with abnormally high or low rates of apoptosis. The presentinvention satisfies this need by providing methods for theadministration of relaxin agonists or antagonists to treatrelaxin-associated tissue abnormalities by modulating apoptosis in suchtissues.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

The present invention relates to the discovery that relaxin isassociated with the development of body tissues. Knockouts of the geneencoding relaxin result in various abnormalities in the development ofvarious body tissues, including smaller size of, increased collagendeposition in, and immaturity of such tissues. Conversely,relaxin-responsive cell accumulation leads to abnormalities in bodytissues. The present invention provides methods of modulating apoptosisin tissues by administering a relaxin agonist or a relaxin antagonist.

In one aspect, the present invention provides methods for modulatingapoptosis in a subject by administering to a subject in need thereof aneffective amount of a relaxin agonist for a period of time sufficient todecrease apoptosis in cells expressing a relaxin receptor. Tissues whichare typically affected by the administration of the relaxin agonistuseful in methods according to the present invention include, forexample, liver, kidney, spleen, thymus, brain, heart, intestine, skin,lung, the male reproductive tract, and the female reproductive tract.Male reproductive tract tissues include, for example, prostatic,epididymal, seminiferous tissues, tissues of the testes, and the like.Female productive tract tissues include, for example, uterus, cervix,the interpubic ligament, connective tissues within the pelvic girdle,and the like.

In certain embodiments, the administration of the relaxin agonisttypically reduces the number of apoptotic cells. In other embodiments,the relaxin agonist stimulates maturation of the tissue. For example, arelaxin agonist can stimulate maturation of male reproductive tracttissue, such as prostatic tissue, epididymal tissue, seminiferoustissue, testicular tissue or sperm. In an embodiment, the maturationresults in an increase in cell number in an under-developed testes, suchas, for example, an increase in the number of mature testicular cells.In another embodiment, maturation of male reproductive tract tissueresults in an increase in the number of viable sperm cells, as comparedwith tissue not contacted with the relaxin agonist. In yet anotherembodiment, fibrosis can be reduced by the relaxin agonist, and/orexcessive collagen deposition can be reduced.

The subject in need of administration of the relaxin agonist can have arelaxin-deficient condition, such as, for example, immature tissue,excessive collagen deposition and/or a low sperm count. For example, theimmature tissue can be immature male productive tissue (e.g.,underdeveloped testes). Such immature tissue can be present in anotherwise mature or immature animal.

The relaxin agonist can be relaxin, a relaxin analog, a small moleculerelaxin effector or a relaxin nucleic acid. The relaxin is typicallyvertebrate relaxin and more typically is human relaxin.

Compositions comprising a relaxin agonist useful in the methodsaccording to the present invention can be formulated for administration,for example, by infusion, injection, oral delivery, nasal delivery aswell as intrapulmonary, rectal, transdermal, interstitial orsubcutaneous delivery. Such compositions also can be formulated fordelayed release of the relaxin agonists into the tissues and circulationof the subject. Particular embodiments comprise compositions formulatedfor infusion or injection, or by intrapulmonary, subcutaneous ortransdermal delivery. In certain embodiments, nucleic acids encodingrelaxin agonists can formulated for administration to the subject in avector encoding the relaxin agonist. For example, the vector can be anexpression vector which expresses the relaxin agonist in the cells.Alternatively, relaxin or relaxin analog nucleic acids can be formulatedfor delivery to the subject. The subjects can be pre-pubescent orpost-pubescent.

In another aspect, the present invention provides methods for modulatingapoptosis in a subject in need thereof by administering an effectiveamount of a relaxin antagonist for a period of time sufficient toincrease apoptosis in a cell population expressing a relaxin receptor.In an embodiment, the relaxin antagonist inhibits binding of relaxin torelaxin receptor. In another embodiment, the relaxin antagonist reducesrelaxin-associated tissue remodeling.

Tissues which are typically affected by the administration of therelaxin antagonist useful in methods according to the present inventioninclude, for example, liver, kidney, spleen, thymus, brain, heart,intestine, skin, lung, the male reproductive tract, the femalereproductive tract, and the like. Male reproductive tract tissuesinclude, for example, prostatic, epididymal, seminiferous tissues,tissues of the testes, and the like. In certain embodiments, the malereproductive tract tissue can be prostatic tissue, and can be mature orimmature. Suitable target female productive tract tissues include, forexample, uterus, cervix, the interpubic ligament, connective tissueswithin the pelvic girdle, and the like. Alternatively, the cellpopulation can comprise cells expressing a relaxin receptor such as, forexample, fibroblasts, osteoblasts, monocytes epithelial cells,endothelial cells, and the like.

The relaxin antagonist can be, for example, a relaxin binding agent, arelaxin receptor binding agent, a relaxin antisense nucleic acid, andthe like. The relaxin binding agent can be, for example, an anti-relaxinantibody, a soluble relaxin receptor, a small molecule relaxinantagonist, and the like. The relaxin receptor binding agent can be, forexample, an anti-relaxin receptor antibody, a relaxin analog, a smallmolecule relaxin receptor antagonist, and the like. Antibodies that bindrelaxin or relaxin receptor can include, for example, polyclonalantibodies, monoclonal antibodies, an Fab, Fab′, an F(ab′)2, an Fv, asingle heavy chain, a chimeric antibody, and the like.

Compositions comprising a relaxin antagonist useful in the methods ofthe present invention can be formulated for administration, for example,by infusion, injection, oral delivery, nasal delivery as well asintrapulmonary, rectal, transdermal, interstitial or subcutaneousdelivery. Compositions can also be formulated for delayed release of therelaxin antagonist into the tissues and circulation of the subject.Particular embodiments comprise compositions formulated for infusion orinjection or by intrapulmonary, subcutaneous or transdermal delivery.

In certain embodiments, nucleic acids encoding relaxin antagonists canalso be administered to the subject in a vector encoding the relaxinantagonist. In one embodiment, the vector is an expression vector whichexpresses the relaxin antagonist in the cell population. Alternatively,relaxin or relaxin receptor antisense nucleic acids can be delivereddirectly to the subject, according to any of the methods describedabove. In a typical embodiment, administration of the relaxin antagonistincreases apoptosis to reduce unwanted cell accumulation. For example,the unwanted cells can be hyperplasia, hypertrophy, cancer or neoplasia.

DEFINITIONS

Prior to setting forth the invention in more detail, it may be helpfulto a further understanding of the invention to set forth definitions ofcertain terms as used hereinafter.

The term “nucleic acid” refers to a polymer composed of a multiplicityof nucleotide units (ribonucleotide or deoxyribonucleotide or relatedstructural variants) linked via phosphodiester bonds. A nucleic acid canbe of substantially any length, typically from about six (6) nucleotidesto about 109 nucleotides, or larger. Unless otherwise stated, theconventional notation used herein for nucleic acids is as follows: theleft-hand end of single-stranded nucleic acid is the 5′ end; theleft-hand direction of double-stranded nucleic acid is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction. Sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences;” sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the coding RNA transcriptare referred to as “downstream sequences”.

Nucleic acids include RNA, mRNA, cDNA, genomic DNA, synthetic forms, andmixed polymers, both sense and antisense strands, and can also bechemically or biochemically modified or can contain non-natural orderivatized nucleotide bases, as will be readily appreciated by thoseskilled in the art. Such modifications include, for example, labels,methylation, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such asuncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoamidates, and carbamates), charged linkages (e.g.,phosphorothioates and phosphorodithioates), pendent moieties (e.g.,polypeptides), intercalators (e.g., acridine and psoralen), chelators,alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).Also included are synthetic molecules that mimic nucleic acids in theirability to bind to a designated sequence via hydrogen bonding and otherchemical interactions. Such molecules are known in the art and include,for example, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

The terms “amino acid” or “amino acid residue”, as used herein, refer toL amino acids or to D amino acids as described further below. Thecommonly used one- and three-letter abbreviations for amino acids areused herein (see, e.g., Alberts et al., Molecular Biology of the Cell,Garland Publishing, Inc., New York (3d ed. 1994)).

The term “conservative substitution,” when describing a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the polypeptide's activity. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, and the like) such that thesubstitution of even critical amino acids does not substantially alteractivity. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), which is incorporated by reference herein.) In addition,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence are also “conservative substitutions.”

The term “relaxin analog” refers to a modified relaxin polypeptide thatincreases or decreases the functional activity of the molecule or itsinteraction with a relaxin receptor.

The term “soluble,” in the context of a polypeptide, refers to theability of the polypeptide to be dissolved in (i.e., to be molecularlyor ionically dispersed in) an aqueous solution, such as water, blood orplasma.

The term “small molecule effector,” in the context of relaxin or arelaxin receptor, refers to an agent that binds to a relaxin, or to arelaxin receptor, and stimulates the activity of relaxin or relaxinreceptor.

The term “small molecule antagonist,” in the context of a relaxin or arelaxin receptor, refers to an agent that binds to a relaxin, or to arelaxin receptor, and reduces or inhibits the activity of relaxin orrelaxin receptor.

The term “effective amount” means a dosage sufficient to provideamelioration of a symptom or treatment for an abnormality, such as adisease, disorder or condition, being treated. The dosage will varydepending on the subject, the abnormality being treated, and thetreatment being effected.

The terms “subnormal,” “subnormally” and “underdeveloped,” in thecontext of body tissue and/or cells, refer to a smaller size, decreasedcell number, and/or immature developmental state, as compared with thesize, cell number and/or developmental state of a tissue and/or cells ofa normal subject of the same age and species of the subject.

The term “tissue” refers to a population of cells, generally consistingof cells of the same kind that perform the same, or a similar, function.A tissue can be part of an organ or bone or it can be a looseassociation of cells, such as cells of the immune system.

The term “maturation,” in the context of body tissue, refers to thedevelopmental progression and/or differentiation of the tissue towardsits full developed state.

The term “mature,” in the context of body tissue, refers to theachievement of full development, differentiation and/or growth of thetissue.

The term “immature,” in the context of body tissue, refers to a tissuethat has not fully developed and/or differentiated.

The term “pre-pubescent male” refers to a male that has not completedthe process of puberty.

The term “post-pubescent male” refers to a male that has completed theprocess of puberty.

The term “puberty” refers to the sequence of events by which a childbecomes a young adult, characterized by the beginning of gametogenesis,secretion of gonadal hormones, development of secondary sexualcharacteristics, and reproductive function.

The term “accumulation,” in the context of body tissue, refers to anincrease in the number of normal (i.e., non-mutant, non-malignant) cellsin the tissue.

The terms “biologically active” and “functionally active” refer to theability of a molecule (e.g., a relaxin agonist or antagonist) to bind toa relaxin or relaxin receptor and to stimulate or inhibit apoptosis,cell accumulation/ and/or tissue maturation, in a body tissue.

The term “relaxin-deficient condition” refers to a disease, disorder orcondition of a subject, in which relaxin levels, or relaxin-receptorlevels, in the relevant tissue, cell(s), or in the subject, are belownormal.

The term “relaxin-responsive” refers to an increase in cell number, orin the state of maturity (i.e., tissue development and/ordifferentiation), in response to the binding of relaxin to a relaxinreceptor.

The term “relaxin-associated” refers to a property, condition orresponse of a cell or tissue by which the cell or tissue is affected,directly or indirectly, by an increase or decrease in functionallyactive relaxin levels, or in functionally active relaxin receptor.

The terms “hyperplasia” or “hyperplastic tissue” refer to an increase inthe number of cells in a tissue.

The terms “hypertrophy” or “hypertrophic” refer to an increase in thesize of a tissue.

The terms “cancer” and “malignancy” generally refer to the various typesof malignant neoplasms, most of which invade surrounding tissues.

The terms “cancerous” and “malignant” relate to cells or tissue havingproperties of cancer or a malignancy.

The term “tissue remodeling” refers to the formation of new cells ortissues and the destruction of existing cells through the apoptoticpathway, in response to the signals mediated by relaxin or arelaxin-receptor.

The term “apoptosis” refers to a regulated network of biochemical eventswhich lead to a selective form of cell suicide, and is characterized byreadily observable morphological and biochemical phenomena, such as thefragmentation of the deoxyribonucleic acid (DNA), condensation of thechromatin, which may or may not be associated with endonucleaseactivity, chromosome migration, margination in cell nuclei, theformation of apoptotic bodies, mitochondrial swelling, widening of themitochondrial cristae, opening of the mitochondrial permeabilitytransition pores and/or dissipation of the mitochondrial protongradient.

DETAILED DESCRIPTION

The present invention provides methods for the administration of relaxinagonists or antagonists for the modulation of apoptosis. The relaxinagonists and antagonists can be used to reduce, manage, treat or preventrelaxin-associated abnormalities. Relaxin-associated abnormalitiesinclude diseases, disorder or conditions of a subject that areassociated with increased or decreased levels of apoptosis, as comparedwith levels of apoptosis in a normal subject. Relaxin-associatedabnormalities include, for example, relaxin-deficient conditions andrelaxin-responsive conditions.

In one aspect, a relaxin agonist is administered to a subject to treat arelaxin-associated abnormality having increased apoptosis, as comparedwith comparable cells from a normal subject (i.e., not having therelaxin-associated abnormality). Such relaxin-associated abnormalitiescan include, for example, immature body tissue, increased collagendeposition, fibrosis (i.e., the presence of abnormal amounts of fibroustissue, as compared with normal tissue), low tissue weight, increasedapoptosis of cells in a cell population in a tissue, and the like.Relaxin-associated abnormalities are typically associated with decreasedlevels of relaxin, and/or relaxin receptor, as compared with normal cellor tissues.

In certain embodiments, the relaxin-associated abnormality is arelaxin-deficient condition, such as, for example, immature tissue, thepresence of excessive collagen, a low sperm count, and the like. Theimmature tissue can be, for example, immature male productive tracttissue (e.g., immature prostatic, epididymal, seminiferous or testiculartissue or sperm). Immature tissue can be characterized, for example, byincreased collagen deposition, low weight, and/or decreased cell number,as compared with a comparable sample of normal tissue. Such tissue alsocan be characterized by its lack of organization, by incompletedevelopment and/or by a lack of, or incomplete, differentiation, ascompared with a comparable sample of normal tissue. In certain otherembodiments, fibrosis can be reduced, such as, for example, dermalfibrosis, lung fibrosis, kidney fibrosis, or age-related fibrosis ofthese or other organs or tissues.

The relaxin agonist is administered in an amount effective to reduce,manage, treat or prevent the relaxin-associated abnormality. Forexample, administration of a relaxin agonist can decrease apoptosis oftarget cells, stimulate maturation, development and/or differentiationof immature tissue, increase tissue weight, increase cell number in thetissue, decrease in collagen deposition, and the like.

In another aspect according to the present invention, a relaxinantagonist is administered to a subject to reduce, manage, treat orprevent a relaxin-associated abnormality by increasing apoptosis in thetarget cells. Such relaxin associated abnormalities can be, for example,relaxin-associated cell accumulation, decreased apoptosis relative tonormal cells or tissue, hyperplasia, hypertrophy, cancer, neoplasia, andthe like. In certain embodiments, the relaxin antagonist can reducerelaxin-associated tissue remodeling, reduce unwanted cell accumulation,reduce or prevent hyperplasia, hypertrophy, cancer, neoplasia, and thelike.

Body tissues having relaxin-associated or relaxin-responsive tissueabnormalities can include, for example, tissues of the brain, heart,liver, kidney, spleen, thymus, intestine, skin, lung, male reproductivetract (e.g., prostate, epididymis, seminal vesicles or testes), orfemale reproductive tract (e.g., the uterus, cervix, the interpubicligament or connective tissues of the pelvic girdle).

Relaxin Agonists

In one aspect according to the present invention, a relaxin agonist isadministered to a subject to treat a relaxin-associated abnormality. Therelaxin agonist can be, for example, a relaxin, a relaxin analog, asmall molecule relaxin effector, a relaxin nucleic acid, and the like.

Relaxin and Relaxin Analogs

In one aspect of the invention, the relaxin agonist is a relaxinpolypeptide or a fragment or analog of a relaxin polypeptide. The term“relaxin” refers to vertebrate relaxin polypeptides, including fulllength relaxin polypeptide or a portion of the relaxin polypeptide thatretains biological activity.

Relaxin has been well defined in its natural human form, animal form,and in its synthetic form. In particular, relaxin has been extensivelydescribed in U.S. Pat. Nos. 5,166,191 and 4,835,251 (both of which arehereby incorporated by reference). In this application, “relaxin”generally refers to the terms “relaxin,” “human relaxin,” “nativerelaxin,” and “synthetic relaxin” as defined in U.S. Pat. No. 5,166,191and the terms “human relaxin” and “human relaxin analogs” as defined inU.S. Pat. No. 4,835,251. In a typical embodiment, the relaxin is humanrelaxin, as described in, for example, U.S. Pat. Nos. 5,179,195;5,023,321; and 4,758,516 (the disclosures of which are incorporated byreference herein). “Relaxin” in this application will also refer torelaxin as isolated in pigs, rats, horses, or other mammalian orvertebrates, and relaxin produced by recombinant techniques using cDNAclones for rat, porcine or other mammalian or vertebrate relaxin(s).

Methods of making relaxin and its analogs are known in the art. Inaddition, methods for isolating and purifying relaxin are known in theart. Several sources for these methods are identified in U.S. Pat. No.5,166,191, including the following references: U.S. Pat. No. 4,835,251,Barany et al., The Peptides 2:1 (1980), Treager et al., Biology ofRelaxin and its Role in the Human, pp. 42-55; EP 0 251 615; EP 0 107782; EP 0 107 045; and WO 90/13659 (all of which are incorporated byreference herein).

Additional methods of making relaxin are described in U.S. Pat. No.5,464,756, and PCT/US94/06997 (the disclosures of which are incorporatedby reference herein). Relaxin can also be prepared by synthesis of the Aand B chains, and purification and assembly thereof, as described inEuropean Patent 0 251 615 (published Jan. 7, 1988, the disclosure ofwhich is incorporated herein by reference). For in vitro assembly ofrelaxin, a 4:1 molar ratio of A to B chains is generally employed. Theresulting product is then purified by any means known to one of ordinaryskill in the art, including, for example, reverse-phase HPLC, ionexchange chromatography, gel filtration, dialysis, and the like, or anycombination of such procedures. Unprocessed or partially processed formsof relaxin, such as preprorelaxin or prorelaxin, can also be used.

In specific embodiments, relaxin polypeptides include the H1 and H2forms of human relaxin. It has been reported that the predominantspecies of human relaxin is the H2 relaxin form with a truncated B chain(i.e., relaxin H2(B29 A24)), wherein the four C-terminal amino acids ofthe B-chain are absent so that the B-chain ends with a serine atposition 29. Either this form (referred to as designated “short relaxin”or “long relaxin,” which contains a B chain of 33 amino acids) can beused.

Relaxin agonists further includes analogs, such as naturally-occurringamino acid sequence variants of relaxin. Relaxin analogs also includethose altered by substitution, addition or deletion of one or more aminoacid residues that provide for functionally active relaxin polypeptides.Such relaxin analogs include, but are not limited to, those containingas a primary amino acid sequence all or part of the amino acid sequenceof a relaxin polypeptide, including altered sequences in which one ormore functionally equivalent amino acid residues are substituted forresidues within the sequence, resulting in a silent functional change(e.g., a conservative substitution).

In another aspect, the relaxin agonist is a polypeptide consisting of orcomprising a fragment of a relaxin polypeptide having at least 10contiguous amino acids of the relaxin polypeptide. Alternatively, thefragment contains at least 20 or 25 contiguous amino acids of therelaxin polypeptide. In other embodiments, the fragments are not largerthan 20 or 30 amino acids.

The relaxin analog can be a polypeptide comprising regions that aresubstantially similar to a relaxin polypeptide or fragments thereof(e.g., in various embodiments, at least 60%, 70%, 75%, 80%, 90%, or even95% identity or similarity over an amino acid sequence of identicalsize), or when compared to an aligned sequence in which the alignment isdone by a computer sequence comparison/alignment program known in theart, or which coding nucleic acid is capable of hybridizing to a relaxinnucleic acid, under high stringency, moderate stringency, or lowstringency conditions (infra). (See, e.g., Smith and Waterman, Adv.Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol. 48:443(1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988);GAP, BESTFIT, FASTA, and TEASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.);Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed.,John Wiley and Sons, New York (1999); the disclosures of which areincorporated by reference herein). Relaxin agonists further comprisefunctionally active relaxin polypeptides, analogs or fragments that bindto a relaxin receptor.

Relaxin agonists, such as relaxin polypeptides, analogs and fragmentscan be produced by various methods known in the art. The manipulationswhich result in their production can occur at the gene or polypeptidelevel. For example, cloned relaxin nucleic acids can be modified by anyof numerous strategies known in the art (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring HarborLaboratory Press, New York (2001); Ausubel et al., Current Protocols inMolecular Biology, 4th ed., John Wiley and Sons, New York (1999); whichare incorporated by reference herein), such as making conservativesubstitutions, deletions, insertions, and the like. The sequence can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of the relaxin nucleic acids encoding an analogor fragment, the modified nucleic acid typically remains in the propertranslational reading frame, so that the reading frame is notinterrupted by translational stop signals or other signals thatinterfere with the synthesis of the relaxin analog or fragment. Therelaxin nucleic acid can also be mutated in vitro or in vivo to createand/or destroy translation initiation and/or termination sequences. Therelaxin nucleic acid can also be mutated to create variations in codingregions and/or to form new restriction endonuclease sites or destroypreexisting ones and to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, chemical mutagenesis, in vitro site-directed mutagenesis(see, e.g., Hutchison et al., J. Biol. Chem. 253:6551-60 (1978)), theuse of TAB® linkers (Pharmacia), and the like. (See generally Sambrooket al., supra; Ausubel et al., supra.)

In a specific embodiment, relaxin analogs are prepared fromrelaxin-encoding nucleic acids that are altered to introduce asparticacid codons at specific position(s) within at least a portion of therelaxin coding region. (See, e.g., U.S. Pat. No. 5,945,402, thedisclosure of which is incorporated by reference herein.) The resultinganalogs can be treated with dilute acid to release a desired analog,thereby rendering the protein more readily isolated and purified. Otherrelaxin analogs are disclosed in U.S. Pat. Nos. 4,656,249; 5,179,195;5,945,402; 5,811,395; and 5,795,807 (the disclosures of which areincorporated by reference herein).

Manipulations of the relaxin polypeptide sequence can also be made atthe polypeptide level. Included within the scope of the invention arerelaxin polypeptides, analogs or fragments that are differentiallymodified during or after synthesis (e.g., in vivo or by in vitrotranslation). Such modifications include conservative substitution,glycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule, another polypeptide or other cellular ligand, and thelike. Any of numerous chemical modifications can be carried out by knowntechniques, including, but not limited to, specific chemical cleavage(e.g., by cyanogen bromide), enzymatic cleavage (e.g., by trypsin,chymotrypsin, papain, V8 protease, and the like); modification by, forexample, NaBH₄, acetylation, formylation, oxidation and reduction,metabolic synthesis in the presence of tunicamycin, and the like.

Relaxin polypeptides, analogs and fragments can be purified from naturalsources by standard methods such as those described herein (e.g.,immunoaffinity purification). Relaxin polypeptides, analogs andfragments can also be isolated and purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, sizing columnchromatography, high pressure liquid chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides. Relaxin polypeptides can be synthesized bystandard chemical methods known in the art (see, e.g., Hunkapiller etal., Nature 310:105-11 (1984); Stewart and Young, Solid Phase PeptideSynthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford, Ill., (1984); thedisclosures of which are incorporated by reference herein).

In addition, analogs of relaxin polypeptides can be chemicallysynthesized. For example, a peptide corresponding to a fragment of arelaxin polypeptide, which comprises a desired domain, or which mediatesa desired activity in vivo, can be synthesized by use of chemicalsynthetic methods using, for example, an automated peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the relaxinpolypeptide sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, 2-amino butyric acid, ε-amino hexanoic acid,6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C α-methyl aminoacids, N α-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In another embodiment, the relaxin agonist is a chimeric, or fusion,protein comprising a relaxin polypeptide, or fragment thereof (typicallyconsisting of at least a domain or motif of the relaxin polypeptide, orat least 10 contiguous amino acids of the relaxin polypeptide), joinedat its amino- or carboxy-terminus via a peptide bond to an amino acidsequence of a different protein. In one embodiment, such a chimericprotein is produced by recombinant expression of a nucleic acid encodingthe chimeric polypeptide. The chimeric product can be made by ligatingthe appropriate nucleic acid sequences, encoding the desired amino acidsequences, to each other in the proper reading frame and expressing thechimeric product by methods commonly known in the art. Alternatively,the chimeric product can be made by protein synthetic techniques (e.g.,by use of an automated peptide synthesizer).

In a specific embodiment, the fusion protein is a relaxin-ubiquitinfusion protein. For example, U.S. Pat. No. 5,108,919 (the disclosure ofwhich is incorporated herein by reference) discloses methods forpreparing a fusion protein of a relaxin chain and ubiquitin.

In preferred embodiments, the relaxin analog, or fragment isfunctionally active (i.e., capable of exhibiting one or more functionalactivities associated with a full-length, wild-type relaxinpolypeptide). As one example, analogs or fragments that retain a desiredrelaxin property of interest (e.g., binding to a relaxin binding partner(e.g., relaxin receptor) and/or modulation (e.g., inhibition) ofapoptosis) can be used as inducers of such property and itsphysiological correlates. A specific embodiment relates to a relaxinanalog or fragment that bind to a relaxin receptor and induces arelaxin-associated decrease in apoptosis. Analogs or fragments ofrelaxin can be tested for the desired activity by procedures known inthe art, including but not limited to the functional assays describedherein.

Relaxin Nucleic Acids:

The invention provides relaxin nucleic acid sequences for expression ofrelaxin polypeptide, fragments and analogs in vivo or in vitro. Therelaxin nucleic acid can be a vertebrate or mammalian relaxin,including, for example, human, mouse, rat, pig, cow, dog, or monkeyrelaxin. The relaxin nucleic acids can comprise genomic nucleic acids,cDNA, the relaxin coding region or a fragment thereof. Relaxin nucleicacids further include mRNAs corresponding to the relaxin locus. Relaxinnucleic acids can also include analogs (e.g., nucleotide sequencevariants), such as those encoding other possible codon choices for thesame amino acid or conservative amino acid substitutions thereof, suchas naturally occurring allelic variants. Due to the degeneracy ofnucleotide coding sequences, other nucleic acid sequences that encodesubstantially the same amino acid sequence as a relaxin cDNA or openreading frame, can be used in the practice of the present invention.These nucleic acid sequences include, but are not limited to, nucleicacid sequences comprising all or portions of a relaxin gene which isaltered by the substitution of different codons that encode the same ora functionally equivalent amino acid residue (e.g., a conservativesubstitution) within the sequence, thus producing a silent change.

The invention further provides relaxin nucleic acid fragments of atleast 6 contiguous nucleotides (e.g., a hybridizable portion); in otherembodiments, the nucleic acids comprise at least 8 contiguousnucleotides, at least contiguous 25 nucleotides, at least contiguous 50nucleotides, at least 100 nucleotides, or at least, 150 nucleotides, ormore of a relaxin sequence. In another embodiment, the nucleic acids aresmaller than 150 nucleotides in length. The relaxin nucleic acids can besingle or double-stranded. As is readily apparent, as used herein, a“nucleic acid encoding a fragment of a relaxin polypeptide” is construedas referring to a nucleic acid encoding only the recited fragment orportion of the relaxin polypeptide and not the other contiguous portionsof the relaxin polypeptide as a contiguous sequence. Fragments ofrelaxin nucleic acids encoding one or more relaxin domains are alsoprovided.

Relaxin nucleic acids, or a functionally active analog or fragmentthereof, can be inserted into an appropriate vector, such as anexpression vector (i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted polypeptide-codingsequence). The necessary transcriptional and translational signals canalso be supplied by the native relaxin gene and/or its flanking regions.A variety of host-vector systems can be utilized to express the relaxinnucleic acid sequences. These include but are not limited to, mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,adeno-associated virus, and the like), insect cell systems infected withvirus (e.g., baculovirus), microorganisms such as yeast containing yeastvectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA,or cosmid DNA. The expression elements of vectors vary in theirstrengths and specificities. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements can be used. In specific embodiments, human relaxin nucleicacids, or a nucleic acid sequence encoding a functionally active portionof human relaxin, is expressed in yeast or bacteria. In yet anotherembodiment, a fragment of relaxin comprising a domain of the relaxinpolypeptide is expressed.

Any of the methods known in the art for the insertion of nucleic acidsinto a vector can be used to construct expression vectors containing achimeric gene consisting of appropriate transcriptional/translationalcontrol signals and relaxin nucleic acid sequences. These methodsinclude in vitro recombinant DNA and synthetic techniques and in vivorecombinants (genetic recombination). Expression of nucleic acidsequence encoding a relaxin polypeptide, analog or fragment can beregulated by a second nucleic acid sequence so that the relaxinpolypeptide, analog or fragment is expressed in a host transformed withthe recombinant DNA molecule. For example, expression of a relaxinpolypeptide can be controlled by any promoter/enhancer element known inthe art. Promoters which can be used to control relaxin gene expressioninclude, but are not limited to, the SV40 early promoter region (Benoistand Chambon, Nature 290:304-10 (1981)), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-97 (1980)), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441-45 (1981)), the regulatory sequencesof the metallothionen gene (Brinster et al., Nature 296:39-42 (1982)),prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-31 (1978)) orthe tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25(1983)), plant expression vectors including the cauliflower mosaic virus35S RNA promoter (Gardner et al., Nucl. Acids Res. 9:2871-88 (1981)),the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., Nature 310:115-20 (1984)),promoter elements from yeast or other fungi such as the Gal7 and Gal4promoters, the ADH (alcohol dehydrogenase) promoter, the PGK(phosphoglycerol kinase) promoter, the alkaline phosphatase promoter,and the like.

The following animal transcriptional control regions, which exhibittissue specificity, have been utilized in transgenic animals: theelastase I gene control region which is active in pancreatic acinarcells (e.g., Swift et al., Cell 38:639-46 (1984); Ornitz et al., ColdSpring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7(1 Suppl.):42S-51S (1987)); the insulin gene control regionwhich is active in pancreatic beta cells (e.g., Hanahan, Nature315:115-22 (1985)), the immunoglobulin gene control region which isactive in lymphoid cells (e.g., Grosschedl et al., Cell 38:647-58(1984); Adams et al., Nature 318:533-38 (1985); Alexander et al., Mol.Cell. Biol. 7:1436-44 (1987)), the mouse mammary tumor virus controlregion which is active in testicular, breast, lymphoid and mast cells(e.g., Leder et al., Cell 45:485-95 (1986)), the albumin gene controlregion which is active in liver (e.g., Pinkert et al., Genes Dev.1:268-76 (1987)), the alpha-fetoprotein gene control region which isactive in liver (e.g., Krumlauf et al., Mol. Cell. Biol. 5:1639-48(1985); Hammer et al., Science 235:53-58 (1987)); the alpha1-antitrypsin gene control region which is active in the liver (e.g.,Kelsey et al., Genes and Devel. 1:161-71 (1987)); the beta-globin genecontrol region which is active in myeloid cells (e.g., Magram et al.,Nature 315:338-40 (1985); Kollias et al., Cell 46:89-94 (1986)); themyelin basic protein gene control region which is active inoligodendrocyte cells in the brain (e.g., Readhead et al., Cell48:703-12 (1987)); the myosin light chain-2 gene control region which isactive in skeletal muscle (e.g., Shani, Nature 314:283-86 (1985)); andthe gonadotropic releasing hormone gene control region which is activein the hypothalamus (e.g., Mason et al., Science 234:1372-78 (1986)). Ina preferred embodiment, the tissue specific promoter is the prostatespecific antigen promoter. (See, e.g., U.S. Pat. No. 6,100,444, thedisclosure of which is incorporated by reference herein.)

In another embodiment, a vector is used that comprises a promoteroperably linked to a relaxin nucleic acid, one or more origins ofreplication, and, optionally, one or more selectable markers (e.g., anantibiotic or drug resistance marker). For example, an expressionconstruct can be made by subcloning a relaxin nucleic acid into arestriction site of the pRSECT expression vector. Such a constructallows for the expression of a relaxin polypeptide, analog or fragmentunder the control of the T7 promoter with a histidine amino terminalflag sequence for affinity purification of the expressed polypeptide. Inanother specific embodiment, a vector is used that comprises theprostate specific antigen promoter operably linked to a relaxin nucleicacid, one or more origins of replication, and, optionally, one or moreselectable markers (e.g., an drug resistance marker).

Expression vectors containing relaxin nucleic acids can be identified bygeneral approaches well known to the skilled artisan, including: (a)nucleic acid hybridization, (b) the presence or absence of “marker” genefunction, (c) expression of inserted sequences, (d) by polymerase chainreaction (PCR), and the like. In the first approach, the presence of arelaxin nucleic acid inserted in an expression vector can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to an inserted relaxin nucleic acid. In the second approach,the recombinant vector/host system can be identified and selected basedupon the presence or absence of certain “marker” gene functions (e.g.,thymidine kinase activity, resistance to antibiotics, transformationphenotype, occlusion body formation in baculovirus, colorimetric change,and the like) caused by the insertion of the relaxin nucleic acids intothe vector. For example, if the relaxin nucleic acid is inserted withinthe marker gene sequence of the vector, recombinants containing therelaxin nucleic acid can be identified by the absence of the marker genefunction.

In the third approach, recombinant expression vectors can be identifiedby assaying the relaxin polypeptide, analog or fragment expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of the relaxin polypeptide, analog or fragment inin vitro assay systems (e.g., binding with anti-relaxin antibody,binding to relaxin receptor, and the like). In a forth approach,recombinant expression vectors can be identified by polymerase chainreaction. (See, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,889,818;Gyllensten et al., Proc. Natl. Acad. Sci. USA 85:7652-56 (1988); Ochmanet al., Genetics 120:621-23 (1988); Loh et al., Science 243:217-20(1989).) Once a particular recombinant vector is identified andisolated, several methods that are known in the art can be used topropagate it.

Once a suitable host system and growth conditions are established,recombinant vectors can be propagated and prepared in quantity. Aspreviously explained, the vectors which can be used include, but are notlimited to the following vectors or their analogs: human or animalviruses such as vaccinia virus or adenovirus; insect viruses such asbaculovirus; yeast vectors; bacteriophage vectors (e.g., lambda); andplasmid and cosmid DNA vectors; to name but a few.

In addition, a host cell strain can be chosen that modulates theexpression of the inserted nucleic acids, or modifies or processes therelaxin, relaxin analog or fragment in the specific fashion desired.Expression from certain promoters can be elevated in the presence ofcertain inducers; thus, expression of the relaxin polypeptide, analog orfragment can be controlled. Furthermore, different host cells havingcharacteristic and specific mechanisms for the translational andpost-translational processing, and modification (e.g., glycosylationand/or phosphorylation) can be used. Appropriate cell lines or hostsystems can be chosen to ensure the desired modification and processingof the polypeptide, analog or fragment expressed. For example,expression in a bacterial system can be used to produce an unprocessedcore protein product. Expression in mammalian cells can be used toensure “native” processing of mammalian relaxin polypeptides, or ofanalogs or fragments. Furthermore, different vector/host expressionsystems can affect processing reactions to different extents.

Relaxin Antagonists

In another aspect of the invention, relaxin antagonists are provided forthe modulation of apoptosis. Relaxin antagonists can include, forexample, relaxin binding agents, relaxin receptor binding agents,antisense nucleic acids, and the like.

Relaxin Antibodies

Relaxin antagonists can comprise antibodies thatimmunospecifically-recognize relaxin or a relaxin receptor polypeptideand that stimulate apoptosis, and/or reduce or inhibitrelaxin-associated cell accumulation in cell populations or tissues.Anti-relaxin and anti-relaxin receptor antibodies include, but are notlimited to, polyclonal antibodies, monoclonal antibodies, chimericantibodies (e.g., fully humanized antibodies or human chimericantibodies), single chain antibodies, antibody fragments (e.g., Fab,F(ab′), F(ab′)₂, Fv, or hypervariable regions), single heavy chains, andan Fab expression library. In a specific embodiment, polyclonal and/ormonoclonal antibodies to full length, vertebrate or mammalian relaxin orrelaxin receptor polypeptide are produced and selected for thoseantibodies that selectively bind to relaxin or a relaxin receptorpolypeptides, and thereby functionally inactivate such polypeptides. Inanother embodiment, antibodies to a domain of a vertebrate relaxinpolypeptide, or a relaxin receptor polypeptide, are produced. In stillanother embodiment, fragments of a vertebrate relaxin polypeptide, or arelaxin receptor polypeptide, which are identified as hydrophilic, areused as immunogens for antibody production and selected forimmunospecific binding to such a polypeptide and inhibition of itsbiological activity.

Various procedures known in the art can be used for the production ofpolyclonal antibodies to a relaxin or relaxin receptor polypeptide, or afragment or analog thereof. For the production of such antibodies,various host animals (including, but not limited to, rabbits, mice,rats, sheep, goats, and the like) can be immunized by injection with thenative relaxin or relaxin receptor polypeptide, or a fragment or analogthereof. Alternatively, transgenic animals having a human immune systemcan be immunized by injection with the native relaxin or relaxinreceptor polypeptide. (See, e.g., U.S. Pat. Nos. 6,114,598 and6,111,166, which are incorporated by reference herein.) Variousadjuvants can be used to increase the immunological response, dependingon the host species, including but not limited to Freund's adjuvant(complete or incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a relaxin orrelaxin receptor polypeptide, fragment, or analog thereof, any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture can also be used. Such techniques include, forexample, the hybridoma technique originally developed by Kohler andMilstein (Nature 256:495-97 (1975)), as well as the trioma technique,the human B-cell hybridoma technique (see, e.g., Kozbor et al.,Immunology Today 4:72 (1983)), and the EBV-hybridoma technique toproduce human monoclonal antibodies (see, e.g., Cole et al., InMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96(1985)). Mammalian antibodies can be used and can be obtained by usinghybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA80:2026-30 (1983)) or by transforming human B cells with EBV virus invitro (see, e.g., Cole et al. (1985), supra). Selection of hybridomasproducing antibodies with appropriate biological function are well knownin the art or are described herein below. Human monoclonal antibodiescan also be prepared by preparing hybridomas from animals having a humanimmune system that have been immunized by injection with the nativerelaxin or relaxin receptor polypeptide. (See, e.g., U.S. Pat. No.6,114,598 and U.S. Pat. No. 6,111, 166, which are incorporated byreference herein.)

Further to the invention, “chimeric” or “humanized” antibodies (see,e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-55 (1984);Neuberger et al., Nature 312:604-08 (1984); Takeda et al., Nature314:452-54 (1985)) can be prepared. Such chimeric antibodies aretypically prepared by splicing the non-human genes for an antibodymolecule specific for a relaxin or receptor polypeptide together withgenes from a human antibody molecule of appropriate activity. It can bedesirable to transfer the antigen binding regions (e.g., an F(ab′)₂,F(ab′), Fv, or hypervariable region(s)) of non-human antibodies into theframework of a human antibody by recombinant DNA techniques to produce asubstantially human molecule. In a preferred embodiment, the antibodiesare fully humanized.

Methods for producing such “chimeric” molecules are generally well knownand described in, for example, U.S. Pat. Nos. 4,816,567; 4,816,397;5,693,762; 5,712,120; 5,821,337; 6,054,297; International PatentPublications WO 87/02671 and WO 90/00616; and European PatentPublication EP 0 239 400 (the disclosures of which are incorporated byreference herein). Alternatively, a human monoclonal antibody orportions thereof can be identified by first screening a human B-cellcDNA library for DNA molecules that encode antibodies that specificallybind to a relaxin or a relaxin receptor polypeptide according to themethod generally set forth by Huse et al. (Science 246:1275-81 (1989)).The DNA molecule can then be cloned and amplified to obtain sequencesthat encode the antibody (or binding domain) of the desired specificity.Phage display technology offers another technique for selectingantibodies that bind to relaxin or relaxin receptor polypeptides,fragments or analogs thereof. (See, e.g., International PatentPublications WO 91/17271 and WO 92/01047; and Huse et al., supra.)

According to another aspect of the invention, techniques for theproduction of single chain antibodies (see, e.g., U.S. Pat. Nos.4,946,778 and 5,969,108) can be adapted to produce relaxin- or relaxinreceptor-specific single chain antibodies. (See also Riechmann andMuyldermans, J. Immunol. Methods 231:25-38 (1999); Muyldermans andLauwereys, J. Mol. Recognit. 12:131-40 (1999)).

An additional aspect of the invention utilizes the techniques describedfor the construction of a Fab expression library (see, e.g., Huse et al.(1989), supra) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for relaxin polypeptides,fragments, or analogs thereof and biological activity.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include, butare not limited to, an F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule, Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, Fabfragments which can be generated by treating the antibody molecule withpapain and a reducing agent, and Fv fragments. Recombinant Fv fragmentscan also be produced in eukaryotic cells using, for example, the methodsdescribed in U.S. Pat. No. 5,965,405.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., ELISA(enzyme-linked immunosorbent assay)). In one example, antibodies whichrecognize a specific domain of a relaxin or relaxin receptor polypeptidecan be used to assay hybridomas for a product (e.g., antibody) thatbinds to a relaxin or relaxin receptor fragment containing that domain.For selection of an antibody that specifically binds to a first relaxinor relaxin receptor polypeptide, but which does not specifically bind asecond, different relaxin polypeptide, one can select on the basis ofantibody-positive binding to the first polypeptide and a lack ofantibody binding to the second, different polypeptide.

Soluble Relaxin Receptors

In another aspect of the invention, the relaxin antagonist is a relaxinbinding agent comprising a soluble relaxin receptor, or a fragment oranalog thereof, that binds relaxin. The term “soluble relaxin receptor”refers to a relaxin receptor polypeptide that is not bound to a cellmembrane. The relaxin receptor is approximately 200 kilodaltons. (SeePalejwala et al., Endocrinology 139(3):1208-12 (1998), the disclosure ofwhich is incorporated by reference herein.) The soluble form of therelaxin receptor retains the ability to bind vertebrate relaxin, buttypically lacks transmembrane and/or cytoplasmic domains. Solublerelaxin receptors can comprise additional amino acid residues, such asaffinity tags, that provide for a means for purification of thepolypeptide or to provide sites for attachment of the polypeptide toanother polypeptide, or to immunoglobulin sequences.

The soluble relaxin receptor can optionally contain a transmembranedomain that cannot associate with a cell membrane. By “transmembranedomain” is meant a domain of the relaxin receptor polypeptide thatcontains a sufficient number of hydrophobic amino acids to allow thepolypeptide to insert and anchor in a cell membrane. By “transmembranedomain that cannot associate with a cell membrane” is meant atransmembrane domain that has been altered by mutation or deletion suchthat it is not sufficiently hydrophobic to allow insertion or otherassociation with a cell membrane. Such a transmembrane domain does notpreclude, for example, the fusion of the relaxin receptor polypeptide,or fragment thereof, with a secretion signal sequence useful forsecretion of the polypeptide from the cell. Substitutions or alterationsof the amino acid sequence useful to achieve an inactive transmembranedomain include, but are not limited to, deletion or substitution ofamino acids within the transmembrane domain. Methods of making solublereceptors are known in the art. (See, e.g., U.S. Pat. Nos. 6,033,903;6,037,450; and 5,925,549; the disclosures of which are incorporated byreference herein.)

The soluble relaxin receptors include soluble, naturally-occurring aminoacid sequence variants of relaxin receptor. Soluble relaxin receptorsfurther include those altered by substitution, addition or deletion ofone or more amino acid residues that provide for functionally activerelaxin receptor polypeptides. Such relaxin receptors include, but arenot limited to, those containing as a primary amino acid sequence of allor part of the amino acid sequence of a relaxin receptor polypeptideincluding sequences in which one or more functionally equivalent aminoacid residues are substituted for residues within the sequence,resulting in a silent functional change (e.g., a conservativesubstitution).

In another aspect, the soluble relaxin receptor is a polypeptideconsisting of or comprising a fragment of a relaxin receptor polypeptidehaving at least 10 contiguous amino acids of the relaxin receptorpolypeptide. More typically, the fragment contains at least 20 or atleast 50 contiguous amino acids of the relaxin receptor polypeptide. Inother embodiments, the fragments are larger than 100 or even 200 aminoacids.

The relaxin receptor polypeptide can be a polypeptide comprising regionsthat are substantially similar to a relaxin receptor polypeptide orfragments thereof (e.g., in various embodiments, at least 60%, 70%, 75%,80%, 90%, or even 95% identity or similarity over an amino acid sequenceof identical size), or when compared to an aligned sequence in which thealignment is done by a computer sequence comparison/alignment programknown in the art, or by visual inspection. (See, e.g., Smith andWaterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol.Biol. 48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988); GAP, BESTFIT, FASTA, and TEASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.); Ausubel et al. (supra); the disclosures of which areincorporated by reference herein). Sequence identity or similarity canalso be determined by identifying nucleic acids that are capable ofhybridizing to a relaxin receptor nucleic acid, under high stringency,moderate stringency, or low stringency conditions. (See, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold SpringHarbor Laboratory Press, New York (2001); Ausubel et al., (1996), supra;the disclosures of which are incorporated by reference herein).

Soluble relaxin receptors and fragments thereof can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,cloned relaxin receptor nucleic acids can be modified by any of numerousstrategies known in the art (see, e.g., Sambrook et al., supra; Ausubelet al., supra), such as making conservative substitutions, deletions,insertions, and the like. The sequence can be cleaved at appropriatesites with restriction endonuclease(s), followed by further enzymaticmodification if desired, isolated, and ligated in vitro. In theproduction of the relaxin receptor nucleic acids, the modified nucleicacid typically remains in the proper translational reading frame, sothat the reading frame is not interrupted by translational stop signalsor other signals which interfere with the synthesis of the solublerelaxin receptor or fragment thereof. The relaxin receptor nucleic acidcan also be mutated in vitro or in vivo to create and/or destroytranslation initiation and/or termination sequences. The relaxinreceptor nucleic acid can also be mutated to create variations in codingregions (e.g., amino acid substitutions) and/or to form new restrictionendonuclease sites or destroy preexisting ones and to facilitate furtherin vitro modification. Any technique for mutagenesis known in the artcan be used, including but not limited to, chemical mutagenesis, invitro site-directed mutagenesis (see, e.g., Hutchison et al., J. Biol.Chem. 253:6551-60 (1978)), the use of TAB® linkers (Pharmacia), and thelike.

Manipulations of the relaxin receptor polypeptide sequence can also bemade at the polypeptide level. Included within the scope of theinvention are relaxin receptor polypeptides that are differentiallymodified during or after synthesis (e.g., in vivo or by in vitrotranslation). Such modifications include conservative substitution,glycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule, a protein or other cellular ligand, and the like. Anyof numerous chemical modifications can be carried out by knowntechniques, including, but not limited to, specific chemical cleavage(e.g., by cyanogen bromide), enzymatic cleavage (e.g., by trypsin,chymotrypsin, papain, V8 protease, and the like); modification by, forexample, NaBH₄, acetylation, formylation, oxidation and reduction,metabolic synthesis in the presence of tunicamycin, and the like.

Relaxin receptor polypeptides and fragments thereof can be purified fromnatural sources by standard methods such as those described herein(e.g., immunoaffinity purification). Relaxin receptor polypeptides andfragments can also be isolated and purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, sizing columnchromatography, high pressure liquid chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides. Relaxin receptor polypeptides andfragments thereof can be synthesized by standard chemical methods knownin the art (see, e.g., Hunkapiller et al., Nature 310:105-11 (1984);Stewart and Young, Solid Phase Peptide Synthesis, 2^(nd) Ed., PierceChemical Co., Rockford, Ill., (1984)). Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the relaxin receptorpolypeptide sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, 2-amino butyric acid, ε-amino hexanoic acid,6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, α-alanine, selenocysteine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C α-methyl aminoacids, N α-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In another embodiment, the soluble relaxin receptor is a chimeric, orfusion, protein comprising a relaxin receptor polypeptide, or fragmentthereof (typically consisting of at least a domain or motif of therelaxin receptor polypeptide, or at least 10 contiguous amino acids ofthe relaxin receptor polypeptide) joined at its amino- orcarboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding thechimeric polypeptide. The chimeric product can be made by ligating theappropriate nucleic acid sequences, encoding the desired amino acidsequences, to each other in the proper reading frame and expressing thechimeric product by methods commonly known in the art. Alternatively,the chimeric product can be made by protein synthetic techniques (e.g.,by use of an automated peptide synthesizer). In a specific embodiment,the fusion protein is a relaxin-receptor-ubiquitin fusion protein.

Relaxin Analogs

The relaxin antagonist can further be a relaxin analog, such as arelaxin polypeptide that binds to a relaxin receptor but fails to inducea response by that receptor. For example, the relaxin analog can be acompetitive inhibitor of relaxin binding or a conventional antagonist ofthe relaxin receptor. The term “relaxin” refers to vertebrate relaxinpolypeptides, including full length relaxin polypeptide or a portion ofthe relaxin polypeptide that retains biological activity. Relaxin hasbeen well defined in its natural human form, animal form, and in itssynthetic form. In particular, relaxin has been extensively described inU.S. Pat. Nos. 5,179,195; 5,166,191; 5,023,321; 4,835,251; and 4,758,516(the disclosures of which are hereby incorporated by reference). Methodsof making relaxin and its analogs are known in the art (supra). Relaxinanalogs can be prepared by modification of relaxin polypeptides suchthat the relaxin analog retains relaxin receptor binding activity, butdoes not induce a response by the relaxin receptor. For example, relaxinanalogs can be amino acid sequence variants of relaxin that retainrelaxin receptor binding activity, but that fail to induce a response bya relaxin receptor. Relaxin analogs further include relaxinpolypeptides, altered by addition or deletion of one or more amino acidresidues, that retain receptor-binding function but fail to induce aresponse by relaxin receptor.

In various aspects according to the present invention, the relaxinanalog is fragment of a relaxin polypeptide consisting of or comprisingat least 10 contiguous amino acids of the relaxin polypeptide.Alternatively, the fragment contains at least 20 or 40 contiguous aminoacids of the relaxin polypeptide. In other embodiments, the fragmentsare not larger than 35 amino acids.

The relaxin analog can be a polypeptide comprising regions that aresubstantially similar to a relaxin polypeptide (e.g., in variousembodiments, at least 60%, 70%, 75%, 80%, 90%, or even 95% identity orsimilarity over an amino acid sequence of identical size), or whencompared to an aligned sequence in which the alignment is done by acomputer sequence comparison/alignment program known in the art, orwhich coding nucleic acid is capable of hybridizing to a relaxin nucleicacid, under high stringency, moderate stringency, or low stringencyconditions. (See supra.)

Relaxin analogs can be produced by various methods known in the art. Themanipulations which result in their production can occur at the gene orprotein level. For example, cloned relaxin nucleic acids can be modifiedby any of numerous strategies known in the art (see, e.g., Sambrook etal., supra; Ausubel et al., supra), such as by making conservative ornon-conservative substitutions, deletions, insertions, and the like. Thesequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification, if desired,isolated, and ligated in vitro. In the production of the relaxin analognucleic acids, the modified nucleic acid typically remains in the propertranslational reading frame, so that the reading frame is notinterrupted by translational stop signals or other signals whichinterfere with the synthesis of the relaxin analog. The relaxin nucleicacid can be mutated in vitro or in vivo to create and/or destroytranslation, initiation and/or termination sequences. The relaxinnucleic acid can also be mutated to create variations in coding regionsand/or to form new restriction endonuclease sites or destroy preexistingones and to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (see, e.g.,Hutchison et al., J. Biol. Chem. 253:6551-60 (1978)), the use of TAB®linkers (Pharmacia), and the like.

In a specific embodiment, relaxin analogs are prepared fromrelaxin-encoding nucleic acids that are altered to introduce asparticacid codons at specific position(s) within at least a portion of therelaxin coding region. (See, e.g., U.S. Pat. No. 5,945,402, thedisclosure of which is incorporated by reference herein.) The resultinganalogs can be treated with dilute acid to release a desired analog,thereby rendering the protein more readily isolated and purified.

Manipulations of the relaxin polypeptide sequence can also be made atthe polypeptide level. Included within the scope of the invention arerelaxin analogs that are differentially modified during or aftersynthesis (e.g., in vivo or by in vitro translation). Such modificationsinclude amino acid substitution (either conservative ornon-conservative), glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule, a protein orother cellular ligand, and the like. Any of numerous chemicalmodifications can be carried out by known techniques, including, but notlimited to, specific chemical cleavage (e.g., by cyanogen bromide),enzymatic cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease,and the like); modification by, for example, NaBH₄, acetylation,formylation, oxidation and reduction, metabolic synthesis in thepresence of tunicamycin, and the like.

Relaxin analogs can be purified from natural sources by standard methodssuch as those described herein (e.g., immunoaffinity purification).Relaxin analogs can also be isolated and purified by standard methodsincluding chromatography (e.g., ion exchange, affinity, sizing columnchromatography, high pressure liquid chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides. Relaxin analogs can be synthesized bystandard chemical methods known in the art (see, e.g., Hunkapiller etal., Nature 310:105-11 (1984); Stewart and Young, Solid Phase PeptideSynthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford, Ill., (1984)).Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the relaxinpolypeptide sequence. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, 2-amino butyric acid, ε-amino hexanoic acid,6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C α-methyl aminoacids, N α-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In another embodiment, the relaxin analog is a chimeric, or fusion,protein comprising a relaxin polypeptide (typically consisting of atleast a domain or motif of the relaxin polypeptide, or at least 10contiguous amino acids of the relaxin polypeptide) joined at its amino-or carboxy-terminus via a peptide bond to an amino acid sequence of adifferent protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding thechimeric polypeptide. The chimeric product can be made by ligating theappropriate nucleic acid sequences, encoding the desired amino acidsequences, to each other in the proper reading frame and expressing thechimeric product by methods commonly known in the art. Alternatively,the chimeric product can be made by protein synthetic techniques (e.g.,by use of an automated peptide synthesizer).

In a specific embodiment, the fusion protein is a relaxinanalog-ubiquitin fusion protein. For example, U.S. Pat. No. 5,108,919(the disclosure of which is incorporated herein by reference), disclosesmethods for preparing a fusion protein of a relaxin chain and ubiquitin.

Nucleic Acids:

The invention further provides nucleic acids for use as an antagonist,or for expressing an antagonist, according to the present invention.Such nucleic acids include those encoding a soluble relaxin receptor ora relaxin analog for synthesis of soluble relaxin receptor or relaxinanalog, respectively. Antisense nucleic acids are provided forinhibition of relaxin or relaxin receptor expression. Nucleic acidsencoding a relaxin polypeptide or a relaxin receptor polypeptide arealso provided for the preparation of antibodies (see supra).

In one aspect, the invention provides nucleic acid sequences encoding arelaxin receptor or relaxin analog for expression in vivo. The relaxinreceptor or relaxin analog, or antisense nucleic acids, can be expressedin vivo for gene therapy. Relaxin receptor, relaxin analog or antisensenucleic acids can also be expressed in vivo or in vitro for theproduction of recombinant soluble relaxin receptor, relaxin analog orantisense nucleic acids for exogenous administration to a subject.

The nucleic acids can be vertebrate nucleic acid, including, forexample, human, mouse, rat, pig, cow, dog, or monkey relaxin receptor,relaxin, or a relaxin analog derived from a vertebrate relaxin. Thenucleic acids can comprise genomic DNA, cDNA, or the coding region ofthe relaxin receptor, relaxin or a relaxin analog. The nucleic acids canfurther include mRNAs corresponding to the relaxin receptor locus or therelaxin locus. The nucleic acids also include nucleotide sequencevariants, such as those encoding other possible codon choices for thesame amino acid or conservative amino acid substitutions thereof, suchas naturally occurring allelic variants. Due to the degeneracy ofnucleotide coding sequences, other nucleic acid sequences which encodesubstantially the same amino acid sequence as a relaxin receptor orrelaxin coding sequence, can be used in the practice of the presentinvention. These nucleic acid sequences include, but are not limited to,nucleotide sequences comprising all or portions of a relaxin gene whichis altered by the substitution of different codons that encode the sameor a functionally equivalent amino acid residue (e.g., a conservativesubstitution) within the sequence, thus producing a silent change.

The invention further provides nucleic acid fragments of at least 6contiguous nucleotides (e.g., a hybridizable portion); in otherembodiments, the nucleic acids comprise at least 8 contiguousnucleotides, at least contiguous 25 nucleotides, at least contiguous 50nucleotides, at least 100 nucleotides, 150 nucleotides or more of arelaxin sequence. In another embodiment, the nucleic acids are smallerthan 100 or 150 nucleotides in length. The nucleic acids can be singleor double-stranded. As is readily apparent, as used herein, a nucleicacid encoding a fragment of a relaxin or relaxin receptor polypeptide isconstrued as referring to a nucleic acid encoding only the recitedfragment or portion of the relaxin polypeptide and not the othercontiguous portions of the relaxin receptor or relaxin polypeptide as acontiguous sequence. Fragments of the nucleic acids encoding one or moredomains of relaxin or relaxin receptor are also provided.

Relaxin receptor, relaxin analog or antisense nucleic acids can beinserted into an appropriate vector (e.g., an expression vector whichcontains the necessary elements for the transcription or transcriptionand translation of the inserted polypeptide-coding sequence) in eitherthe sense or antisense orientations, as desired. The necessarytranscriptional and translational signals can also be supplied by thenative relaxin or relaxin receptor gene and/or its flanking regions. Avariety of host-vector systems can be utilized to express thepolypeptide-coding sequence. These include but are not limited to,mammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, adeno-associated virus, and the like), insect cell systemsinfected with virus (e.g., baculovirus), microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophage,DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors varyin their strengths and specificities. Depending on the host-vectorsystem utilized, any of a number of suitable transcription andtranslation elements can be used. In specific embodiments, the nucleicacids are expressed, or a nucleic acid sequence encoding a functionallyactive portion of a relaxin receptor or relaxin analog is expressed inmammalian cells, yeast or bacteria. In yet another embodiment, afragment of a relaxin receptor or relaxin analog comprising a domain ofthe respective polypeptide is expressed.

Any of the methods known in the art for the insertion of nucleic acidsinto a vector can be used to construct expression vectors containing achimeric gene having the appropriate transcriptional, translationalcontrol signals and/or polypeptide coding sequences. These methodsinclude in vitro recombinant DNA and synthetic techniques and in vivorecombinants (genetic recombination). Expression of nucleic acidsencoding a relaxin receptor or relaxin analog can be regulated by asecond nucleic acid sequence so that the nucleic acid or polypeptide isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of a nucleic acid of a relaxin receptor or relaxinanalog can be controlled by any promoter/enhancer element known in theart. Promoters which can be used to control expression include, but arenot limited to, the SV40 early promoter region (see, e.g., Benoist andChambon, Nature 290:304-10 (1981)), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (see, e.g., Yamamoto et al.,Cell 22:787-97 (1980)), the herpes thymidine kinase promoter (see, e.g.,Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-45 (1981)), theregulatory sequences of the metallothionen gene (see, e.g., Brinster etal., Nature 296:39-42 (1982)), prokaryotic expression vectors such asthe β-lactamase promoter (see, e.g., Villa-Komaroff et al., Proc. Natl.Acad. Sci. USA 75:3727-31 (1978)) or the tac promoter (see, e.g., deBoeret al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)), plant expressionvectors including the cauliflower mosaic virus 35S RNA promoter (see,e.g., Gardner et al., Nucl. Acids Res. 9:2871-88 (1981)), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(see, e.g., Herrera-Estrella et al., Nature 310:115-20 (1984)), promoterelements from yeast or other fungi such as the Gal7 and Gal4 promoters,the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerolkinase) promoter, the alkaline phosphatase promoter, and the like.

The following animal transcriptional control regions, which exhibittissue specificity, have been utilized in transgenic animals: theelastase I gene control region which is active in pancreatic acinarcells (see, e.g., Swift et al., Cell 38:639-46 (1984); Ornitz et al.,Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7(1 Suppl.):42S-51S (1987)); the insulin gene control regionwhich is active in pancreatic beta cells (see, e.g., Hanahan, Nature315:115-22 (1985)), the immunoglobulin gene control region which isactive in lymphoid cells (see, e.g., Grosschedl et al., Cell 38:647-58(1984); Adams et al., Nature 318:533-38 (1985); Alexander et al., Mol.Cell. Biol. 7:1436-44 (1987)), the mouse mammary tumor virus controlregion which is active in testicular, breast, lymphoid and mast cells(see, e.g., Leder et al., Cell 45:485-95 (1986)), the albumin genecontrol region which is active in liver (see, e.g., Pinkert et al.,Genes Dev. 1:268-76 (1987)), the alpha-fetoprotein gene control regionwhich is active in liver (see, e.g., Krumlauf et al., Mol. Cell. Biol.5:1639-48 (1985); Hammer et al., Science 235:53-58 (1987)); the alpha1-antitrypsin gene control region which is active in the liver (see,e.g., Kelsey et al., Genes and Devel. 1:161-71 (1987)); the beta-globingene control region which is active in myeloid cells (see, e.g., Magramet al., Nature 315:338-40 (1985); Kollias et al., Cell 46:89-94 (1986));the myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (see, e.g., Readhead et al., Cell48:703-12 (1987)); the myosin light chain-2 gene control region which isactive in skeletal muscle (see, e.g., Shani, Nature 314:283-86 (1985));and the gonadotropic releasing hormone gene control region which isactive in the hypothalamus (see, e.g., Mason et al., Science 234:1372-78(1986)). In a preferred embodiment, the tissue-specific promoter is theprostate specific antigen promoter. (See, e.g., U.S. Pat. No. 6,100,444,the disclosure of which is incorporated by reference herein.)

In another embodiment, a vector is used that comprises a promoteroperably linked to a nucleic acid, one or more origins of replication,and, optionally, one or more selectable markers (e.g., an antibiotic ordrug resistance marker). For example, an expression construct can bemade by subcloning a relaxin receptor or relaxin analog nucleic acidinto a restriction site of the pRSECT expression vector. Such aconstruct allows for the expression of the relaxin analog or relaxinreceptor polypeptide under the control of the T7 promoter with ahistidine amino terminal flag sequence for affinity purification of theexpressed polypeptide. In another specific embodiment, a vector is usedthat comprises the prostate specific antigen promoter operably linked toa relaxin analog nucleic acid, one or more origins of replication, and,optionally, one or more selectable markers (e.g., a drug resistancemarker).

Expression vectors containing such nucleic acids can be identified bygeneral approaches well known to the skilled artisan, including: (a)nucleic acid hybridization or polymerase chain reaction, (b) thepresence or absence of “marker” gene function, (c) expression ofinserted sequences, or polymerase chain reaction (PCR). In the firstapproach, the presence of a nucleic acid inserted in an expressionvector can be detected by nucleic acid hybridization or polymerase chainreaction using probes comprising sequences that are homologous to aninserted nucleic acid. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain “marker” gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, and the like) caused by theinsertion of a vector containing the relaxin receptor, relaxin orrelaxin analog nucleic acids. For example, if the nucleic acid isinserted within the marker gene sequence of the vector, recombinantscontaining the nucleic acid can be identified by the absence of themarker gene function.

In the third approach, recombinant expression vectors can be identifiedby assaying the polypeptide expressed by the recombinant. Such assayscan be based, for example, on the physical or functional properties ofthe polypeptide in in vitro assay systems (e.g., binding withanti-relaxin antibody, binding relaxin or a relaxin analog, binding torelaxin receptor, and the like). Once a particular recombinant vector isidentified and isolated, several methods that are known in the art canbe used to propagate it. Once a suitable host system and growthconditions are established, recombinant expression vectors can bepropagated and prepared in quantity. As previously explained, theexpression vectors which can be used include, but are not limited to thefollowing vectors or their derivatives: human or animal viruses such asvaccinia virus or adenovirus; insect viruses such as baculovirus; yeastvectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmidDNA vectors, to name but a few. In the fourth approach, PCR is used todetect the nucleic acid in the vector (see supra).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies or processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the polypeptide can be controlled. Furthermore, differenthost cells having characteristic and specific mechanisms for thetranslational and post-translational processing, and modification (e.g.,glycosylation, phosphorylation) of polypeptides can be used. Appropriatecell lines or host systems can be chosen to ensure the desiredmodification and processing of the foreign protein expressed. Forexample, expression in a bacterial system can be used to produce anunprocessed core protein product. Expression in mammalian cells can beused to ensure “native” processing of mammalian receptor polypeptide.Furthermore, different vector/host expression systems can affectprocessing reactions to different extents.

Functional Assays for Relaxin and Relaxin Receptor Agonists andAntagonists

The activity of relaxin agonists and antagonists, and of relaxinreceptor agonists and antagonists, can be determined by standard assaysfor relaxin and/or relaxin receptor activity. In one aspect of theinvention, the activity of a relaxin agonist is assayed. For example,the ability of a relaxin agonist to decrease apoptosis, or to stimulatematuration of tissue, is assayed.

In another aspect of the invention, the ability of a relaxin antagonistto inhibit relaxin functional activity by binding to relaxin is assayed.Similarly, the ability of a relaxin antagonist to inhibit relaxinfunction, or relaxin receptor function, can be assayed by, for example,adding a relaxin antagonist to a relaxin receptor assay and determiningthe inhibition, as compared with a control without the relaxinantagonist. Suitable measurements of relaxin antagonist activity includemeasuring percent inhibition, IC₅₀, and the like.

Suitable assays for measuring relaxin or relaxin receptor agonist orantagonist activity, include, for example, those described in thefollowing references (which are incorporated by reference herein):MacLennan et al., Ripening of the Human Cervix and Induction of Laborwith Intracervical Purified Porcine Relaxin, Obstetrics & Gynecology68:598-601 (1986); Poisner et al., Relaxin Stimulates the Synthesis andRelease of Prorenin From Human Decidual Cells: Evidence ForAutocrine/Paracrine Regulation, J. Clinical Endocrinology and Metabolism70:1765-67 (1990); O'Day-Bowman et al., Hormonal Control of the Cervixin Pregnant Gilts. III. Relaxin's Influence on Cervical BiochemicalProperties in Ovariectomized Hormone-Treated Pregnant Gilts,Endocrinology 129:1967-76 (1991); Saugstad, Persistent Pelvic Pain andPelvis Joint Instability, Eur. J. Obstetrics & Gynecology andReproductive Biology 41:197-201 (1991).

Other assays include those disclosed by Bullesbach et al., TheReceptor-Binding Sites of Human Relaxin II, J. Biol. Chem. 267:22957-60(1992); Hall et al., Influence of Ovarian Steroids on Relaxin-InducedUterine Growth in Ovariectomized Gilts, Endocrinology 130:3159-66(1992); Kibblewhite et al., The Effect of Relaxin on Tissue Expansion,Arch. Otolaryngol. Head Neck Surg. 118:153-56 (1992); Lee et al.,Monoclonal Antibodies Specific for Rat Relaxin. VI. Passive Immunizationwith Monoclonal Antibodies Throughout the Second Half of PregnancyDisrupts Histological Changes Associated with Cervical Softening atParturition in Rats, Endocrinology 130:2386-91 (1992); Bell et al., ARandomized, Double-Blind Placebo-Controlled Trial of the Safety ofVaginal Recombinant Human Relaxin for Cervical Ripening, Obstetrics &Gynecology 82:328-33 (1993); Bryant-Greenwood et al., SequentialAppearance of Relaxin, Prolactin and IGFBP-1 During Growth andDifferentiation of the Human Endometrium, Molecular and CellularEndocrinology 95:23-29 (1993); Chen et al., The Pharmacokinetics ofRecombinant Human Relaxin in Nonpregnant Women After Intravenous,Intravaginal, and Intracervical Administration, Pharmaceutical Research10:834-38 (1993); Huang et al., Stimulation of Collagen Secretion byRelaxin and Effect of Oestrogen on Relaxin Binding in Uterine CervicalCells of Pigs, Journal of Reproduction and Fertility 98:153-58 (1993);

Additional assays are disclosed in Saxena et al., Is the Relaxin Systema Target for Drug Development? Cardiac Effects of Relaxin, TiPS 14:231(June 1993, letter); Winn et al., Hormonal Control of the Cervix inPregnant Gilts. IV. Relaxin Promotes Changes in the HistologicalCharacteristics of the Cervix that are Associated with CervicalSoftening During Late Pregnancy in Gilts, Endocrinology 133:121-28(1993); Colon et al., Relaxin Secretion into Human Semen Independent ofGonadotropin Stimulation, Biology of Reproduction 50:187-92 (1994);Golub et al., Effect of Short-Term Infusion of Recombinant Human Relaxinon Blood Pressure in the Late-Pregnant Rhesus Macaque (Macaca Mulatta),Obstetrics & Gynecology 83:85-88 (1994); Jauniaux et al., The Role ofRelaxin in the Development of the Uteroplacental Circulation in EarlyPregnancy, Obstetrics & Gynecology 84:338-342 (1994); Johnson et al.,The Regulation of Plasma Relaxin Levels During Human Pregnancy, J.Endocrinology 142:261-65 (1994); Lane et al., Decidualization of HumanEndometrial Stromal Cells in Vitro: Effects of Progestin and Relaxin onthe Ultrastructure and Production of Decidual Secretory Proteins, HumanReproduction 9:259-66 (1994); Lanzafame et al., PharmacologicalStimulation of Sperm Motility, Human Reproduction 9:192-99 (1994);Petersen et al., Normal Serum Relaxin in Women with Disabling PelvicPain During Pregnancy, Gynecol. Obstet. Invest. 38:21-23 (1994); Tashimaet al., Human Relaxins in Normal, Benign and Neoplastic Breast Tissue,J. Mol. Endocrinology 12:351-64 (1994); Winn et al. Individual andCombined Effects of Relaxin, Estrogen, and Progesterone inOvariectomized Gilts. I. Effects on the Growth, Softening, andHistological Properties of the Cervix, Endocrinology 135:1241-49 (1994);Winn et al., Individual and Combined Effects of Relaxin, Estrogen, andProgesterone on Ovariectomized Gilts. II. Effects on MammaryDevelopment, Endocrinology 135:1250-55 (1994); Bryant-Greenwood et al.,Human Relaxins: Chemistry and Biology, Endocrine Reviews 15:5-26 (1994);Johnson et al., Relationship Between Ovarian Steroids, Gonadotrophinsand Relaxin During the Menstrual Cycle, Acta Endocrinilogica 129:121-25(1993).

In yet another aspect of the invention, the activity of an agonist orantagonist is determined by measuring the ability of the agonist orantagonist to compete with wild-type relaxin polypeptide, or relaxinreceptor polypeptide, for binding to anti-relaxin antibody. Variousimmunoassays known in the art can be used. Such assays include, but arenot limited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay) “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, and the like),Western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays or hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, and the like. Antibody binding can bedetected by measuring the amount of label on the primary antibody thatis bound, or prevented from binding to, a substrate. Alternatively,primary antibody binding is detected by measuring binding of a secondaryantibody or reagent to the primary antibody. The secondary antibody canalso be directly labeled. Many means are known in the art for detectingbinding in an immunoassay and are considered within the scope of thepresent invention.

The functional activity of an agonist or antagonist can also bedetermined in an in vivo system. For example, the ability of relaxinagonists or antagonists to bind, or to compete for binding to, a relaxinreceptor, or to modulate apoptosis in a cell population and/or tissuescan be measured. The assays described above can be used to determine theactivity resulting from expression of relaxin agonist or antagonists invertebrate cells. Alternatively, relaxin agonist or antagonist can beexpressed in a heterologous system and the activity of the relaxinagonist or antagonist can be assayed as a modulator of a physiologicalchange in that system. For example, the ability of a relaxin agonist orantagonist to modulate apoptosis can be tested in vertebrate cells(e.g., transfected mammalian cells).

Administration of Relaxin Agonists and Antagonists

The invention provides methods for the administration to a subject of aneffective amount of a relaxin agonist or antagonist (also referred tocollectively as an “active agent”). Typically, the active agent issubstantially purified prior to formulation. The subject can be a humanor non-human animal, a vertebrate, and is typically an animal, includingbut not limited to, cows, pigs, horses, chickens, cats, dogs, and thelike. More typically, the subject is a mammal, and in a particularembodiment, human.

Various delivery systems are known and can be used to administer aactive agent, such as, for example, by infusion, injection (e.g.,intradermal, intramuscular or intraperitoneal), oral delivery, nasaldelivery, intrapulmonary delivery, rectal delivery, transdermaldelivery, interstitial delivery or subcutaneous delivery. In a specificembodiment, it can be desirable to administer the active agent locallyto the area in need of treatment; this administration can be achievedby, for example, and not by way of limitation, local infusion, topicalapplication, by injection (e.g., intratesticular or intraprostatic), bymeans of a catheter, or by means of an implant, the implant being forexample, a porous, non-porous, gelatinous or polymeric material,including membranes such as silastic membranes or fibers. In oneembodiment, administration can be by direct injection at the targetsite.

Pharmaceutical compositions containing the active agent can beformulated according to the desired delivery system. Such pharmaceuticalcompositions typically comprise a therapeutically effective amount ofactive agent and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in vertebrates,typically animals, and more typically in humans. The term “carrier”refers to a diluent, adjuvant, excipient, stabilizer, preservative,viscogen, or vehicle with which the active agent is formulated foradministration. Pharmaceutical carriers can be sterile liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Suitable excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, ethanol, and the like. The composition, if desired,can also contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

Suitable preservatives include, for example, sodium benzoate, quaternaryammonium salts, sodium azide, methyl paraben, propyl paraben, sorbicacid, ascorbylpalmitate, butylated hydroxyanisole, butylatedhydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine,potassium benzoate, potassium metabisulfite, potassium sorbate, sodiumbisulfite, sulfur dioxide, organic mercurial salts, phenol and ascorbicacid. Suitable viscogens include, for example, carboxymethylcellulose,sorbitol, dextrose, and polyethylene glycols. Other examples of suitablepharmaceutical carriers are described in, for example, Remington'sPharmaceutical Sciences (Gennaro (ed.), Mack Publishing Co., Easton, Pa.(1990)). The active agents can also be formulated as neutral or saltforms.

Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, and the like, and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

In one embodiment, the active agent is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. For intravenous delivery,water is a typical carrier. Saline, aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition can also include a solubilizing agent and alocal anesthetic to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water-freeconcentrate in a hermetically sealed container such as an ampoule orsachet indicating the quantity of active agent. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientscan be mixed prior to administration.

Orally deliverable compositions can take the form of solutions,suspensions, emulsions, tablets, pills, capsules, powders,sustained-release formulations, and the like. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like.

For rectal administration, the compositions are formulated according tostandard pharmaceutical procedures. Typically, the composition is formedas a meltable composition, such as a suppository. Suppositories cancontain adjuvants which provide the desired consistency to thecomposition. They can also contain water-soluble carriers, such aspolyethylene glycol, polypropylene glycol, glycerogelatine,methylcellulose or carboxymethylcellulose. Wetting agents, such as fattyacids, fatty acid glycerides, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylenefatty acid esters, as well as higher alcohol esters of polyoxyethyleneand esters of lower alkylsulfonic acids. The suppositories can alsocontain suitable emulsifying and dispersing components as well ascomponents for adjusting the viscosity and coloring substances.

Nasal administration is typically performed using a solution as a nasalspray and can be dispensed by a variety of methods known to thoseskilled in the art. Systems for intranasally dispensing liquids as aspray are well known (see, e.g., U.S. Pat. No. 4,511,069, which isincorporated by reference herein). Preferred nasal spray solutionscomprise the active agent in a liquid carrier that optionally includes anonionic surfactant for enhancing absorption of the drug and one or morebuffers or other additives to minimize nasal irritation. In someembodiments, the nasal spray solution further comprises a propellant.The pH of the nasal spray solution is typically between about pH 6.8 and7.2.

For intranasal administration, ingredients which improve the absorptionof nasally administered active agent and reduce nasal irritation,especially when used in a chronically administered treatment protocol,are desirable. In this context, the utilization of surfactants toenhance absorption of the active agent is preferred. (See, e.g., Hiraiet al., Int. J. Pharmaceutics 1:173-84 (1981); Great Britain PatentSpecification 1 527 605, each of which is incorporated by referenceherein.) Nasal administration of drugs enhanced by surfactants, however,can cause nasal irritation, including stinging, congestion andrhinorrhea. Thus, compositions which enhance absorption through thenasal mucosa with reduced irritation are desirable, such as, forexample, nonionic surfactants such as nonoxynol-9, laureth-9,poloxamer-124, octoxynol-9 and lauramide DEA. Nonoxynol-9 (N-9) is anethoxylated alkyl phenol, the polyethyleneoxy condensate of nonylphenolwith 9 moles of ethylene oxide. This surfactant has been used indetergent products and is sold under trade names such as SURFONIC® N-95(Jefferson), NEUTRONYX® 600 (Onyx) and IGEPAL® (CO-630 (GAF). N-9 isconsidered to be a hard detergent, and has been used as a spermatocide.(See The Merck Index, 10^(th) Ed., Entry 6518). To minimize irritationattributed to employment of surfactants, one or more anti-irritantadditives are included in the emulsion. In one example, polysorbate-80has been shown to reduce the irritation caused by intranasallyadministered drugs where delivery was enhanced by use of a nonionicsurfactant (see, e.g., U.S. Pat. No. 5,902,789, which is incorporated byreference herein).

Intrapulmonary dosage forms containing the active agent can beadministered to the respiratory tract intranasally or by breathing aspray or aerosol containing the active agent. The active agent istypically delivered directly into the lungs in a small particle aerosol,which is specifically targeted to the smallest air passages and alveoli.

Intrapulmonary dosage forms are typically formed as particulatedispersed forms. This can be accomplished by preparing an aqueousaerosol of solid particles which contain the composition. Typically, anaqueous aerosol is made by formulating an aqueous solution or suspensionof the composition together with conventional pharmaceuticallyacceptable carriers and stabilizers. The carriers and stabilizerstypically include nonionic surfactants (e.g., Tweens, Pluronics orpolyethylene glycol), innocuous proteins such as serum albumin, sorbitanesters, oleic acid, amino acids such as glycine, buffers, salts, sugarsor sugar alcohols. The formulations can also include mucolytic agents,such as those described in U.S. Pat. No. 4,132,803 (which isincorporated by reference herein), as well as broncho-dilating agents.The formulations are preferably sterile. Aerosols are generally preparedfrom isotonic solutions. The particles optionally include normal lungsurfactant proteins.

The aerosol of particles can be formed in aqueous or nonaqueous (e.g.,fluorocarbon propellant) suspensions. The aerosols are preferably freeof lung irritants (i.e., substances that cause acutebronchoconstriction, coughing, pulmonary edema or tissue destruction).Nonirritating, absorption enhancing agents are also suitable for useherein.

Sonic nebulizers can be used to prepare aerosols. Sonic nebulizersminimize exposure of the composition to shear, which can result indegradation. A suitable device is the Bird Micronebulizer. Othersuitable atomizing or nebulizing systems or intratracheal deliverysystems include, for example, those disclosed in U.S. Pat. No.3,915,165; European Patent No. 0 166 476; the jet nebulizers describedby Newman et al. (Thorax 40:671-76 (1985)); metered dose inhalers (see,e.g., Berenberg, J. Asthma-USA 22:87-92 (1985)); the endotrachealcatheter assembly of Braunner (U.S. Pat. No. 5,803,078), or otherdevices (see, e.g., Sears et al., N.Z. Med. J. 96:74311 (1983); O'Reillyet al., Br. Med. J. 286:6377 (1983); or Stander et al., Respiration44:237-40 (1982)), so long as they are compatible with the compositionto be administered and are capable of delivering particles of thedesired size. (The disclosures of these references are incorporated byreference herein.)

The particulate aerosol suspensions are typically fine dry powderscontaining the active agent. Particulate aerosol suspension are preparedby any number of conventional procedures. The simplest method ofpreparing such suspensions is to micronize the active agent (e.g., ascrystals or lyophilization cakes), and suspend the particles in dryfluorocarbon propellants. In these formulations the active agent ispreferably suspended in the fluorocarbon. In an alternate embodiment,the active agent is stored in a compartment separate from thepropellant. Discharge of the propellant withdraws a predetermined dosefrom the storage compartment. The devices used to deliver active agentsin this manner are known as metered dose inhalers (MDIs) (see, e.g.,Byron, Drug Development and Industrial Pharmacy 12:993 (1986), which isincorporated by reference herein).

The size of the aerosols or particles generally will range about from0.5 to about 5 μm, typically about 2 μm to 5 μm, preferably about 2 toabout 4 μm or about 4 to about 5 μm. In some aspects, smaller particlesare less acceptable because they tend not to be deposited, but insteadare exhaled. Similarly, in other aspects, larger particles are notpreferred because they are less likely to be deposited in the alveoli,being removed by impaction within the nasopharyngeal or oral cavities(see, e.g., Byron, J. Pharm. Sci. 75:433 (1986)). The aerosol orparticulate compositions can be heterogeneous in size distribution,although heterogeneity can be reduced by known methods (e.g., thescreening unit described in EP 0 135 390). Heterogeneity is typicallynot disadvantageous unless the proportion of particles having an averagemean diameter in excess of about 4 μm is so large as to impair thedelivery of a therapeutic dose by pulmonary inhalation. Suspensionscontaining greater than about 15% of particles within the 0.5-5 μm rangecan be used, but generally the proportion of particles having an averagemean diameter larger than 5 μm is typically less than about 25%, andpreferably not greater than 10%, of the total number of particles. Thediameters recited refer to the particle diameters as introduced into therespiratory tract.

The amount of the active agent which will be effective in the treatmentof a particular subject will depend on the specific abnormality beingtreated, and can be determined by standard clinical techniques. Inaddition, in vitro assays can optionally be employed to help identifyoptimal dosage ranges. The precise dose of the active agent to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the condition, and should bedecided according to the judgment of the practitioner and each subject'scircumstances. Suitable dosage ranges for administration are generallyabout 0.001 mg/kg to about 100 mg/kg of active agent per kilogram bodyweight. Effective doses can also be extrapolated from dose responsecurves derived from in vitro or animal model test systems. Suppositoriesgenerally contain active ingredient in the range of 0.5% to 10% byweight; oral formulations typically contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In yet another embodiment, the active agent can be delivered in acontrolled release system. In one embodiment, a pump can be used (see,e.g., Langer, supra; Sefton, Crit. Ref Biomed. Eng. 14:201-40 (1987);Buchwald et al., Surgery 88:507-16 (1980); Saudek et al., N. Engl. J.Med. 321:574-79 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howardet al., J. Neurosurg. 71:105-12 (1989)) (the disclosures of which areincorporated by reference herein).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, Medical Applications ofControlled Release, supra, Vol. 2, pp. 115-38 (1984)). Other controlledrelease systems are discussed in, for example, the review by Langer(Science 249:1527-33 (1990), which is incorporated by reference herein).

Administration of Nucleic Acids

The invention provides further methods for the administration of nucleicacids (e.g., nucleic acids encoding relaxin agonists or antagonists,such as relaxins, relaxin analogs, soluble relaxin receptor, relaxinbinding agents, relaxin receptor binding agents, relaxin antisensenucleic acids and/or relaxin receptor antisense nucleic acids) tomodulate apoptosis. Nucleic acids, both sense and antisense, can be usedin the process of gene therapy. Gene therapy refers to the process ofproviding for the expression of nucleic acids of exogenous origin,including antisense nucleic acids or those encoding relaxin agonist orantagonist in a subject for the modulation of apoptosis (e.g. to treat atissue abnormality) within the subject. In specific embodiments, nucleicacids encoding a relaxin or a relaxin analog are administered todecrease apoptosis in cells have a relaxin receptor. In other specificembodiments, nucleic acids encoding a relaxin binding agent (e.g., arelaxin a soluble relaxin receptor) or a relaxin receptor binding agent(e.g., a relaxin analog) is administered to increase apoptosis in a cellpopulation expressing a relaxin receptor. Any of the methods for genetherapy available in the art can be used according to the presentinvention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Steinberg andRaso (J. Pharm. Pharm. Sci. 1:48-59 (1998)); Pantuck et al. (World J.Urol. 18:143-47 (2000)); Prince (Pathology 30:335-47 (1998)); Ledley(Curr. Opin. Biotechnol. 5:626-36 (1994)); Goldspiel et al. (Clin.Pharm. 12:488-505 (1993)); Wu and Wu (Biotherapy 3:87-95 (1991));Tolstoshev (Ann. Rev. Pharmacol. Toxicol. 32:573-96 (1993)); Mulligan(Science 260:926-32 (1993)); Morgan and Anderson (Ann. Rev. Biochem.62:191-217 (1993)); and May (TIBTECH 11:155-215 (1993)).

Methods commonly known in the art of recombinant DNA technology that canbe used include those described in Ausubel et al. (1996, supra) andKriegler (Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990)). In one embodiment, the nucleic acid comprises a sensenucleic acid (e.g., encoding a relaxin analog, soluble relaxin receptor,and the like) that is part of a vector that expresses the nucleic acidin a suitable host. In particular, such a nucleic acid has a promoteroperably linked to the coding region (e.g., relaxin or relaxin receptor)in a sense orientation, the promoter being inducible or constitutive,and, optionally, tissue-specific. In another embodiment, the nucleicacid comprises an antisense nucleic acid (e.g., a relaxin antisensenucleic acid or relaxin receptor antisense nucleic acid) that is part ofa vector that expresses the nucleic acid in a suitable host. Inparticular, such a nucleic acid has a promoter operably linked to thecoding region (e.g., relaxin or relaxin receptor) in an antisenseorientation, the promoter being inducible or constitutive, and,optionally, tissue-specific.

In another particular embodiment, a nucleic acid (e.g., a sense nucleicacid encoding a relaxin analog, soluble relaxin receptor, and the like,or an antisense nucleic acid encoding a relaxin antisense nucleic acid,a relaxin receptor antisense nucleic acid, and the like) used in whichthe nucleic acid and any other desired sequences are flanked by regionsthat promote homologous recombination at a desired site in the genome,thus providing for intrachromosomal expression of the nucleic acid (see,e.g., Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-35 (1989);Zijlstra et al., Nature 342:435-38 (1989); U.S. Pat. Nos. 5,631,153;5,627,059; 5,487,992; and 5,464,764; the disclosures of which areincorporated by reference herein).

For any of these embodiments, delivery of the nucleic acid into asubject can be either direct, in which case the subject is directlyexposed to the nucleic acid or nucleic acid-carrying vector, orindirectly, in which case cells are first transformed with the nucleicacid in vitro, and then transplanted into the subject. These twoapproaches are known, respectively, as in vivo or ex vivo gene therapy.In a specific embodiment, the nucleic acids are directly administered invivo, where they are expressed to produce the encoded product. This canbe accomplished by any of numerous methods known in the art (e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, for example, byinfection using a defective or attenuated retroviral or other viralvector (infra), by direct injection of naked DNA (see, e.g., Asahara etal., Semin. Interv. Cardiol. 1:225-32 (1996); Prazeres et al., TrendsBiotechnol. 17:169-74 (1999); the disclosures of which are incorporatedby reference herein), electroporation (see, e.g., Muramatsu et al., Int.J. Mol. Med. 1:55-62 (1998), the disclosure of which is incorporated byreference herein), or by use of microparticle bombardment, such as agene gun (BIOLISTIC™, Dupont); see, e.g., Biewenga et al., J. Neurosci.Methods 71:67-75 (1997), the disclosure of which is incorporated byreference herein). Nucleic acids can also be inserted into cells bycoating naked nucleic acids with lipids or cell-surface receptors ortransfection agents, encapsulation in liposomes, derivatized liposomes,microparticles, or microcapsules (see, e.g., De Smedt et al., Pharm.Res. 17:113-26 (2000); Maurer et al., Mol. Membr. Biol. 16:129-40(1999); Tarahovsky and Ivanitsky, Biochemistry 63:607-18 (1998); Lasci,Trends Biotechnol. 16: 307-21 (1998); Gao and Huang, Gene Ther. 2:710-22(1995); the disclosures of which are incorporated by reference herein).Nucleic acids can also be administered in linkage to a peptide which isknown to enter the cell or by administering the nucleic acids in linkageto a ligand subject to receptor-mediated endocytosis, which can be usedto target cell types specifically expressing the receptors, and thelike. (See, e.g., Liang et al., Pharmazie 54:559-66 (1999); Cristiano,Front. Biosci. 15:D1161-70 (1998); Guy et al., Mol. Biotechnol. 3:237-48(1995); Wu and Wu, J. Biol. Chem. 262:4429-32 (1987); the disclosures ofwhich are incorporated by reference herein). In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. (See, e.g., Phillips, Biologicals23:13-16 (1995), the disclosure of which is incorporated by referenceherein.)

In yet another embodiment, the antisense nucleic acid can be targeted invivo for cell specific uptake and expression by targeting a specificreceptor (see, e.g., Phillips, Biologicals 23:13-16 (1995);International Patent Publications WO 92/06180; WO 92/22635; WO 92/20316;WO 93/14188, and WO 93/20221; the disclosures of which are incorporatedby reference herein).

In a specific embodiment, a viral vector is used that contains thenucleic acid (e.g., a sense nucleic acid encoding a relaxin analog,soluble relaxin receptor, and the like, or an antisense nucleic acidencoding a relaxin antisense nucleic acid, a relaxin receptor antisensenucleic acid, and the like). For example, a retroviral vector can beused (see, e.g., Palu et al., Rev. Med. Virol. 10:185-202 (2000);Buchschacher and Wong-Staal, Blood 15:2499-504 (2000); Miller et al.,Meth. Enzymol. 217:581-99 (1993), the disclosures of which areincorporated by reference herein). These retroviral vectors aretypically modified to delete retroviral sequences that are not necessaryfor packaging of the viral genome and integration into host cell DNA.The antisense nucleic acid to be used in gene therapy is cloned into thevector, which facilitates delivery of the antisense nucleic acid intothe subject. Lentiviral vectors can also be used. (See, e.g.,Buchschacher and Wong-Staal, supra; Naldini et al., Science 272:263-67(1996), the disclosures of which are incorporated by reference herein).Other references illustrating the use of viral vectors in gene therapyare by Lundstrom (J. Recept. Signal. Transduct. Res. 19:673-86 (1999));Clowes et al. (J. Clin. Invest. 93:644-51 (1994)); Kiem et al. (Blood83:1467-73 (1994)); Salmons and Gunzberg (Hum Gene Ther. 4:129-41(1993)); and Grossman and Wilson (Curr. Opin. Genet Dev. 3:110-14(1993)) (the disclosures of which are incorporated by reference herein).

Adenoviruses can also be used in gene therapy. Adenoviruses areespecially attractive vehicles for delivering genes to prostate, liver,the central nervous system, endothelial cells, and muscle. Adenoviruseshave the advantage of being capable of infecting non-dividing cells.Kozarsky and Wilson (Curr. Opin. Genet Dev. 3:499-503 (1993), thedisclosure of which is incorporated by reference herein) present areview of adenovirus-based gene therapy. Herman et al. (Human GeneTherapy 10:1239-49 (1999), the disclosure of which is incorporated byreference herein) describe the intraprostatic injection of areplication-deficient adenovirus containing the herpes simplex thymidinekinase gene into human prostate, followed by intravenous administrationof the prodrug ganciclovir in a phase I clinical trial. Other instancesof the use of adenoviruses in gene therapy can be found in Rosenfeld etal. (Science 252:431-34 (1991)); Rosenfeld et al. (Cell 68:143-55(1992)); Mastrangeli et al. (J. Clin. Invest. 91:225-34 (1993)); andThompson (Oncol. Res. 11:1-8 (1999)). Adeno-associated virus (AAV) canalso be used in gene therapy (see, e.g., Rabinowitz and Samulski, Curr.Opin. Biotechnol. 9:475-85 (1988); Carter and Samulski, Int. J. Mol.Med. 6:17-27 (2000); Tal, J. Biomed. Sci. 7:279-91 (2000); Ali et al.,Gene Therapy 1:367-84 (1994); U.S. Pat. Nos. 4,797,368 and 5,139,941;Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); Grimm etal., Human Gene Therapy 10:2445-50 (1999)) (the disclosures of which areincorporated by reference herein).

Another approach to gene therapy involves transferring a nucleic acid tocells in tissue culture by methods such as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Typically,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the nucleic acid. Theselected cells are then delivered to a subject.

In one embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid, cell fusion, chromosome-mediated genetransfer, microcell-mediated gene transfer, and the like. Numeroustechniques are known in the art for the introduction of foreign genesinto cells (see, e.g., Muramatsu et al., Int. J. Mol. Med. 1:55-62(1998); Liang et al., Pharmazie 54:559-66 (1999); Loeffler and Behr,Meth. Enzymol. 217:599-618 (1993); Cotten et al., Meth. Enzymol.217:618-44 (1993); Cline, Pharmacol. Ther. 29:69-92 (1985); thedisclosures of which are incorporated by reference herein) and can beused in accordance with the present invention. The technique typicallyprovides for the stable transfer of the nucleic acids to the cell, sothat the nucleic acids are expressible by the cell and is heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Typically, cells are injected subcutaneously.In another embodiment, recombinant skin cells can be applied as a skingraft onto the subject. The amount of cells required for use depends onthe desired effect, the subject's condition, and the like, and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to cells or populations of cells of the male reproductivetract (e.g., prostate cells, cells of the testes, seminiferous cells, orepididymal cells), female reproductive tract (e.g., uterus, cervix, theinterpubic ligament, connective tissues within the pelvic girdle, andthe like), liver, kidney, spleen, thymus, brain, heart, intestine, skin,lung, and the like. Suitable cells further include epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, and stem orprogenitor cells. The cells used for gene therapy generally areautologous to the subject, but heterologous cells that can be typed forcompatibility with the subject can be used.

In another aspect, nucleic acids (e.g., a sense nucleic acid encoding arelaxin analog, soluble relaxin receptor, and the like, or an antisensenucleic acid encoding a relaxin antisense nucleic acid, a relaxinreceptor antisense nucleic acid, and the like) are administered directlyto cells. The nucleic acids are at least six nucleotides and aretypically oligonucleotides (ranging from 6 to about 50 nucleotides ormore). In specific aspects, the oligonucleotide is at least 10nucleotides, at least 15 nucleotides, at least 100 nucleotides, or canbe at least 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or analogs thereof, and can besingle-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide can include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see, e.g.,Nielsen, Pharmacol. Toxicol. 86:3-7 (2000); Soomets et al., Front.Biosci. 1:D782-86 (1999); Galderisi et al., J. Cell Physiol. 181:251-57(1999); Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553-56 (1989);Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-52 (1987);International Patent Publication WO 88/09810), hybridization-triggeredcleavage agents (see, e.g., Krol et al., BioTechniques 6:958-76 (1988))or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539-49 (1988)).(The disclosures of these references are incorporated herein.)

In one embodiment, antisense oligonucleotides are provided assingle-stranded DNA. The oligonucleotides can be modified at anyposition on its structure with substituents generally known in the art.The oligonucleotides can comprise at least one modified base moiety,such as, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine, and the like.In another embodiment, the oligonucleotides comprise at least onemodified sugar moiety, such as, for example, arabinose,2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotides comprise atleast one modified phosphate backbone, such as, for example, aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (see Gautier etal., Nucl. Acids Res. 15:6625-41 (1987)). The oligonucleotide can beconjugated to another molecule (e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, and the like).

In a specific embodiment, the antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., Welch et al., Curr. Opin.Biotechnol. 9:486-96 (1998); Norris et al., Adv. Exp Med. Biol.465:293-301 (2000); International Patent Publication WO 90/11364; Sarveret al., Science 247:1222-25 (1990)). In another embodiment, theoligonucleotide is a 2′-0-methylribonucleotide (Inoue et al., Nucl.Acids Res. 15:6131-48 (1987)), or a chimeric RNA-DNA analogue (Inoue etal., FEBS Lett. 215:327-30 (1987)).

In another specific embodiment, double-stranded RNA directs thesequence-specific degradation of mRNA by RNA interference. (Seegenerally Hunter, Curr. Biol. 10:R137-40 (2000); Bosher and Labouesse,Nat. Cell. Biol. 2:e31-36 (2000); the disclosures of which areincorporated by reference herein.) Briefly, double-stranded nucleicacids are introduced into a cell to selectively inhibit gene expressionby causing degradation of the mRNA. (See, e.g., Zamore et al., Cell101:25-33 (2000), the disclosure of which is incorporated by referenceherein.)

Nucleic acids according to the present invention can be synthesized bystandard methods known in the art. Enzymatic methods for the synthesisof nucleic acids frequently employ Klenow, T7, T4, Taq or Escherichiacoli DNA polymerases, as described in Sambrook et al. (supra). Enzymaticmethods of RNA nucleic acids frequently employ SP6, T3 or T7 RNApolymerases, as described in Sambrook et al. (supra). Reversetranscriptase can also be used to synthesize DNA from RNA (Sambrook etal., supra). Nucleic acids are typically prepared enzymatically using atemplate nucleic acid which can either be synthesized chemically, or beobtained as mRNA, genomic DNA, cloned genomic DNA, cloned cDNA or othernucleic acid. Some enzymatic methods of DNA nucleic acid synthesis canrequire an additional primer which can be synthesized chemically.Finally linear nucleic acids can be prepared by polymerase chainreaction (PCR) techniques as described, for example, by Saiki et al.(Science 239:487 (1988)).

Chemical methods can also be used to synthesize nucleic acids (e.g.,antisense oligonucleotides), such as by use of a commercially availableautomated DNA synthesizer). As examples, phosphorothioate nucleic acidscan be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209-21 (1988)), methylphosphonate nucleic acids can be prepared byuse of controlled pore glass polymer supports (see, e.g., Sarin et al.,Proc. Natl. Acad. Sci. USA 85:7448-51 (1988)) (the disclosures of whichare incorporated by reference herein), and the like. Other methodsinclude those disclosed by Usman et al. (J. Am. Chem. Soc. 109:7845-54(1987)), Scaringe et al. (Nucleic Acids Res. 18:5433-41 (1990));Caruthers (Oligonucleotides: Antisense Inhibitors of Gene Expression,pp. 7-24, Cohen, (ed.), CRC Press, Inc. Boca Raton, Fla., 1989));Oligonucleotide Synthesis, A Practical Approach (Gait (ed.), IRL Press,1984); Oligonucleotides and Analogues, A Practical Approach (Eckstein,IRL Press, 1991); and in U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707;4,668,777; 4,973,679; 5,026,838; 5,132,418; and Re. 34,069. All of theforegoing references are incorporated by reference herein.

Small Molecule Effectors or Antagonists

Relaxin nucleic acids, polypeptides, analogs and fragments, and relaxinreceptor nucleic acids, polypeptides, analogs and fragments and analogs,also have uses in screening assays to detect candidate compounds thatspecifically bind to relaxin polypeptides, or to relaxin receptor, andmodulate apoptosis. Such candidate compounds are typically smallmolecule effectors (agonists) or antagonists, and can be identified byin vitro and/or in vivo assays. Such assays can be used to identifysmall molecule effectors or antagonists that are therapeuticallyeffective as relaxin agonists or antagonists or as lead compounds fordrug development. The invention thus provides assays to detect compoundsthat specifically affect the activity or expression of relaxin nucleicacids, relaxin polypeptides, relaxin receptor nucleic acids, relaxinreceptor, and the like.

In a typical in vivo assay, recombinant cells expressing relaxin orrelaxin receptor nucleic acids can be used to screen candidate compoundsfor those that affect relaxin or relaxin receptor nucleic acidexpression. Agonistic or antagonistic effects on relaxin or relaxinreceptor expression can include stimulation or inhibition (e.g., up ordown regulation) of transcription of mRNA, a increase or decrease inmRNA stability, translation of the mRNA, synthesis of relaxin or relaxinreceptor polypeptides, relaxin or relaxin receptor polypeptide function(e.g., binding to relaxin receptor), and/or effects on relaxin orrelaxin receptor polypeptide stability or localization. Such effects onexpression can be identified as physiological changes, such as, forexample, changes in relaxin-responsive tissue growth rate, division,viability, collagen deposition, apoptosis, and the like. In oneembodiment, candidate compounds are administered to recombinant cellsexpressing a relaxin or relaxin receptor polypeptide to identify thosecompounds that produce a physiological change (e.g., stimulate orinhibit relaxin or relaxin receptor polypeptide function).

Candidate compounds can also be identified by in vitro screeningmethods. For example, recombinant cells expressing a relaxin or arelaxin receptor nucleic acid can be used to recombinantly producerelaxin or relaxin receptor polypeptide for in vitro assays to identifycandidate compounds that bind to relaxin or relaxin receptorpolypeptide. Candidate compounds (such as small molecules) are contactedwith the polypeptide (or a fragment or analog thereof) under conditionsconducive to binding, and then candidate compounds that specificallybind to the polypeptide are identified. Methods that can be used tocarry out the foregoing are commonly known in the art, and includediversity libraries, such as random or combinatorial peptide ornon-peptide libraries that can be screened for candidate compounds thatspecifically bind to relaxin or relaxin receptor polypeptide. Manylibraries are known in the art that can be used, for example, includechemically synthesized libraries, recombinant phage display libraries,and in vitro translation-based libraries.

Examples of chemically synthesized libraries are described in Fodor etal. (Science 251:767-73 (1991)), Houghten et al. (Nature 354:84-86(1991)), Lam et al. (Nature 354:82-84 (1991)), Medynski (BioTechnology12:709-10 (1994)), Gallop et al. (J. Med. Chem. 37(9):1233-51 (1994)),Ohlmeyer et al. (Proc. Natl. Acad. Sci. USA 90:10922-26 (1993)), Erb etal. (Proc. Natl. Acad. Sci. USA 91:11422-26 (1994)), Houghten et al.(BioTechniques 13:412-21 (1992)), Jayawickreme et al. (Proc. Natl. Acad.Sci. USA 91:1614-18 (1994)), Salmon et al. (Proc. Natl. Acad. Sci. USA90:11708-12 (1993)), International Patent Publication WO 93/20242, andBrenner and Lerner (Proc. Natl. Acad. Sci. USA 89:5381-83 (1992)). (Thedisclosures of these references are incorporated herein.)

Examples of phage display libraries are described in Scott and Smith(Science 249:386-90 (1990)), Devlin et al. (Science 249:404-06 (1990)),Christian et al. (J. Mol. Biol. 227:711-18 (1992)), Lenstra (J. Immunol.Meth. 152:149-57 (1992)), Kay et al. (Gene 128:59-65 (1993)), andInternational Patent Publication WO 94/18318, the disclosures of whichare incorporated by reference herein.

In vitro translation-based libraries include, but are not limited to,those described in International Patent Publication WO 91/05058, andMattheakis et al. (Proc. Natl. Acad. Sci. USA 91:9022-26 (1994)). By wayof examples of nonpeptide libraries, a benzodiazepine library (see,e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91:4708-12 (1994)) can beadapted for use. Peptide libraries (see, e.g., Simon et al., Proc. Natl.Acad. Sci. USA 89:9367-71(1992)) can also be used. Another example of alibrary that can be used, in which the amide functionalities in peptideshave been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (Proc. Natl. Acad.Sci. USA 91:11138-42 (1994)).

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, for example, the following references,which disclose screening of peptide libraries: Parmley and Smith (Adv.Exp. Med. Biol. 251:215-18 (1989)); Scott and Smith (1990, supra);Fowlkes et al. (BioTechniques 13:422-28 (1992)); Oldenburg et al. (Proc.Natl. Acad. Sci. USA 89:5393-97 (1992)); Yu et al. (Cell 76:933-45(1994)); Staudt et al. (Science 241:577-80 (1988)); Bock et al. (Nature355:564-66 (1992)); Tuerk et al. (Proc. Natl. Acad. Sci. USA 89:6988-92(1992)); Ellington et al. (Nature 355:850-52 (1992)); U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346; Rebar and Pabo (Science 263:671-73(1994)); and International Patent Publication WO 94/18318, thedisclosures of which are incorporated by reference herein.

In a specific embodiment, screening can be carried out by contacting thelibrary members with relaxin, a relaxin analog , or a relaxin receptorimmobilized on a solid phase and harvesting those library members thatbind to the relaxin, relaxin analog or relaxin analog. Examples of suchscreening methods, termed “panning” techniques are described by way ofexample in Parmley and Smith (Gene 73:305-18 (1988)); Fowlkes et al.(1992, supra); International Patent Publication WO 94/18318; and inreferences cited herein.

Identifying a Subject with a Relaxin-Associated Abnormality

Relaxin nucleic acids (both sense and antisense), and fragments andanalogs thereof, and anti-relaxin antibodies, also have utility toidentify subjects with a relaxin-associated abnormality. Such moleculescan be used in assays, such as hybridization or immunoassays, to detect,prognose, diagnose, or monitor various abnormalities, to determinewhether relaxin expression, or the response to relaxin or a relaxinanalog is affected. Similarly, such molecules have utility to monitorthe treatment of the cell or tissue abnormalities. In particular,methods, such as an immunoassay, can be carried out by steps comprisingcontacting a sample derived from a subject with an anti-relaxin antibodyunder conditions conducive to immunospecific binding, and detecting ormeasuring the amount of any immunospecific binding of protein by theantibody.

Binding of antibody to relaxin or relaxin receptor polypeptide, intissue sections or from seminal fluid, can be used to detect aberrant(e.g., low, absent or elevated) levels of relaxin and/or relaxinreceptor polypeptide. In a specific embodiment, antibody to relaxin orrelaxin receptor polypeptide can be used to assay a subject's tissue orseminal fluid for the presence of relaxin or relaxin receptorpolypeptide, where an aberrant level of relaxin is an indication of arelaxin-associated abnormality. By “aberrant levels” is meant increasedor decreased levels relative to that present, or to a standard levelrepresenting that present, in an analogous sample from a portion of thebody or from a subject not having the abnormality.

The immunoassays which can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such asWestern blot, radioimmunoassay, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassay, immunoprecipitation assay, precipitinreaction, gel diffusion precipitin reaction, immunodiffusion assay,agglutination assay, complement-fixation assay, immunoradiometric assay,fluorescent immunoassay, protein A immunoassay, and the like.

Relaxin and relaxin receptor nucleic acids (both sense and antisense),including fragments and analogs thereof, can also be used inhybridization assays. Such nucleic acids, comprising or consisting of atleast contiguous 8 nucleotides, can be used as hybridization probes orfor polymerase chain reaction detection. Hybridization assays can beused to detect, prognose, diagnose, or monitor diseases or conditionsassociated with aberrant relaxin or relaxin receptor expression and/oractivity, as described supra. In particular, a hybridization assay canbe carried out by a method comprising contacting a sample containingpolynucleotides with a nucleic acid probe capable of hybridizing torelaxin or relaxin receptor DNA or RNA, under conditions such thathybridization can occur, and detecting or measuring any resultinghybridization.

In a specific embodiment, abnormalities associated with over- orunder-expression of relaxin can be diagnosed, or their suspectedpresence can be screened for, or a predisposition to develop suchabnormalities can be identified by detecting decreased or increasedlevels of relaxin polypeptide, relaxin RNA, or relaxin functionalactivity. Additionally, over-expression of relaxin or increased relaxinfunctional activity can be diagnosed by detecting mutations in relaxinRNA or DNA or relaxin polypeptide (e.g., translocations in relaxinnucleic acids, truncations of the relaxin gene or relaxin polypeptide,changes in the nucleotide or amino acid sequence relative to wild-typerelaxin, respectively) that cause increased expression or activity ofrelaxin polypeptide.

By way of example, levels of relaxin polypeptide in a biopsy or fromseminal fluid can be detected by immunoassay of tissues; levels ofrelaxin RNA can be detected by hybridization assays (e.g., Northern blotor dot blot). Translocations and point mutations in relaxin or relaxinreceptor nucleic acids can be detected by Southern blot, RFLP analysis,PCR using primers that detect point mutations, deletions or insertions,sequencing of the relaxin genomic DNA or cDNA obtained from the sample,and the like.

In another embodiment, levels of relaxin or relaxin receptor mRNA orpolypeptide in a sample of tissue isolated from a subject are detectedor measured, in which increased levels indicate that the subject has, orhas a predisposition to a relaxin-associated tissue abnormality.

In yet another embodiment, abnormal relaxin receptor activity in atissue is detected or measured using any of the functional assaysdescribed above. Abnormal relaxin receptor activity can include, forexample, an increased or decreased number of a relaxin receptors onrelaxin-responsive cells, the presence of relaxin receptors on cellsthat are not normally responsive to relaxin, increased or decreasedrelaxin receptor response time, an increased or decreased bindingaffinity for relaxin or relaxin receptor, or a change in thedissociation constant of relaxin from a relaxin receptor, and the like.

Kits for diagnostic and/or prognostic use are also provided thatcomprise, in one or more containers, a relaxin agonist or antagonistand, optionally, a labeled binding partner to an antibody.Alternatively, an antibody can be labeled with a detectable marker(e.g., a chemiluminescent, enzymatic, fluorescent, a radioactive moiety,and the like). A kit is also provided that comprises, in one or morecontainers, a nucleic acid probe capable of hybridizing to relaxin orrelaxin receptor mRNA or DNA.

In another embodiment, the kit can comprise in one or more containers apair of primers (e.g., each in the size range of 6-30 nucleotides ormore) that are capable of priming amplification (e.g., by polymerasechain reaction (see, e.g., Innis et al., PCR Protocols, Academic Press,Inc., San Diego, Calif. (1989)), ligase chain reaction (see, e.g., EP 0320 308), use of Qβ replicase, cyclic 5′ probe reaction, or othermethods known in the art) under appropriate reaction conditions suchthat at least a portion of a relaxin nucleic acid is amplified. A kitcan optionally further comprise in a container a predetermined amount ofa purified relaxin or relaxin receptor nucleic acid, for example, foruse as a standard or control.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

EXAMPLE 1

The effect of inactivation of one or both alleles of the mouse RLX geneswas examined. The following methods and materials were followed.

Animals

Wild-type, heterozygous and homozygous relaxin knockout mice wereobtained from the Howard Florey Institute of Experimental Physiology andMedicine (Parkville, Victoria, 3052, Australia) and used to establish abreeding program. Subsequent generations of RLX +/+ (wild-type), RLX +/−(heterozygous) and rlx −/− (null mutant) mice were generated from RLX+/− parents. All animals were housed in a controlled environment andmaintained on a 14 hours light, 10 hours dark schedule with access toLabdiet rodent lab chow (Deans Animal Feed, San Bruno, Calif.) andwater. These experiments were approved by the Institute's AnimalExperimental Ethics Committee, which adheres to the NIH Code of Practicefor the care and use of laboratory animals.

Genotyping by PCR

Mouse DNA was isolated by lysing tail tissue (5-7 millimeters) in 400 μlof PCR lysis buffer, containing 50 mM Tris-HCl, pH 8.0, 0.5% SDS, 0.1 MEDTA and 1 mg/ml proteinase K (Gibco BRL, Gaithersburg, Md.) at 50-55°C. overnight. Digested samples were then mixed with 3M sodium acetate(40 μl), buffer saturated phenol (200 μl) and chloroform (200 μl) inserum vaccutainer tubes, before samples were centrifuged at 3000 rpm for10 minutes. The DNA (contained in the upper aqueous phase) was decantedinto separate microcentrifuge tubes containing isopropyl alcohol (240μl) to precipitate the DNA before samples were vortexed, spun and thesupernatant discarded. The remaining DNA pellet was dissolved in 40-50μl of sterile water. For PCR, each DNA template (1 μl) was used in a 30μl reaction mixture containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mMKCl, 1.5 mM MgCl₂), 2.5 mM dNTPs, 2.5 U Taq Polymerase (PGC Scientific,Gaithersburg, Md.) and 150 ng of each of the RLX +/+ and rlx −/−primers, designed by Zhao and colleagues (Zhao Endocrinology 140:445-53(1999)). The amplification protocol consisted of an initial denaturationstep at 94° C. (3 minutes) followed by 35 sequential cycles of 94° C.(60 seconds), 55° C. (60 seconds) and 72° C. (90 seconds) and concludedby an additional 10 minute extension at 72° C. 15 μl of each sample werethen analyzed by electrophoresis on a 2% (w/v) agarose gel, and stainedwith ethidium bromide. A 235 by product was generated by primersdesigned from the wild-type allele, while a 170 by product was generatedfrom the mutant allele primers (Zhao (1999), supra).

Tissue Collection and Histology

Relaxin wildtype, heterozygous and homozygous males and females (n≧20 ineach of the 6 groups) were obtained at 1 week, 1 month, 2 months and 3months of age and weighed. RLX +/+ and rlx −/− male mice were thensacrificed under anesthesia with carbon dioxide for tissue collection.The male reproductive tract (including testis, epididymis, prostate,seminal vesicle and attached fat) were collected from each animal at 1week of age (n=10 RLX +/+ males, n=11 −/− males); 1 month of age (n=10RLX +/+males, n=10 −/− males); and 3 months of age (n=10 RLX +/+ males,n=10 rlx −/− males. After weighing each tissue, they were placed in 10%formalin for histological analysis.

The collected tissues were processed (sequentially dehydrated) from 70%alcohol to paraffin before being embedded and cut (4 pm sections), usingan AO Spencer 820 microtome and placed on poly-L-lysine coated glassslides. Consecutive sections from each tissue were stained with H & E(hematoxylin and eosin) and for collagen, with Masson trichrome(staining kit, Richard-Allan Scientific®, Kalamazoo, Mich.) as describedby the manufacturer. The stained slides were viewed using a ZeissAxioplan-2® microscope, the images captured by digital camera(Hamamatsu) and stored for retrieval and analysis. The images weredigitally enhanced for maximum contrast and brightness using AdobePhotoshop (Adobe Systems® Inc, Mountain View, Calif.).

Antibody Staining of Paraffin-Embedded Tissue Sections

The tissues from the reproductive tract of one and three month old malemice were mounted on precoated slides and deparaffinized by heating at58° C. (about 30 minutes), then washed three times in xylene, twice inabsolute ethanol and twice in 95% ethanol, before being briefly soakedin water. The samples were then stained using an Immunocruz® stainingsystem utilizing a horseradish peroxidase (HRP)-streptavidin complex(Santa Cruz Biotechnology®, Inc, Santa Cruz, Calif.) in a humidifiedatmosphere. Tissue sections were initially treated with a peroxidaseblocker (to quench endogenous peroxidase activity) (5 minutes) beforebeing preblocked in goat serum (20 minutes). Serial sections from eachRLX +/+ and rlx −/− tissue sample were then incubated with either a Baxmonoclonal IgG primary antibody (4 μg/ml) (Santa Cruz Biotechnology®,Inc), a caspase-9 polyclonal IgG antibody (4.5 μg/ml) (Santa CruzBiotechnology®, Inc.) or a proliferating cell nuclear antigen (PCNA)(Santa Cruz Biotechnology® Inc.) monoclonal IgG antibody (4 μg/ml) (2hours). Depending on the type of antibody used, either a mouse IgG orrabbit IgG control (Santa Cruz Biotechnology®, Inc.) stain of tissues (2hours) was also used in all experiments performed. Samples were washedin PBS (2 minutes), subjected to the appropriate secondary antibody(goat anti-mouse IgG or goat anti-rabbit IgG) (30 minutes), washed asabove (2 minutes), treated with a HRP-streptavidin complex (30-45minutes) and incubated with a diaminobenzidine chromagen substrate (2-10minutes) that was prepared in accordance with the manufacturer'sinstructions. The slides were then washed in distilled water (2 minutes)before being dehydrated from 95% alcohol to xylene and mounted, thenphotographed as described above.

Statistical Analysis

The results were analyzed using a one-ANOVA test. All data in this paperare presented as the mean±SEM, with p<0.05 considered statisticallysignificant.

EXAMPLE 2 The Effects of Relaxin Gene Knockout on the Growth Of Mice

The body weights of male and female relaxin wildtype (+/+), heterozygous(+/−) and null mutant (−/−) mice were measured at 1 week, 1 month, 2months and 3 months of age (n=20-21 for each group). No significantdifferences in mean body weight were noted in male (RLX +/+: 3.87±0.09g; rlx −/−: 3.6±0.13 g) or female (RLX +/+: 3.52±0.09 g; rlx −/−:3.37±0.08 g) mice at 1 week of age. However, at the time the mice were 1month of age, mean body weights of both rlx −/− males (17.05±0.65 g) andfemales (14.77±0.42 g) were significantly less (p<0.05) than theirrespective RLX wildtype counterparts (RLX +/+ M: 18.92±0.61 g; RLX +/+F: 16.34±0.51 g). Male and female null mutant mice continued to besignificantly smaller (p<0.05) than RLX wildtype animals at 2 months ofage (RLX +/+ M: 25.42±0.41 g; rlx −/− M: 24.23±0.42 g; RLX +/+ F:20.96±0.32 g; rlx −/− F: 19.94±0.22 g); however, the differences in sizebetween the two groups were less than that observed at 1 month of age.By adulthood (3 months of age), the mean weight of rlx −/− mice (rlx −/−M: 26.57±0.47 g; rlx −/− F: 22.54±0.31 g) was still slightly less thanthe average weight of adult RLX +/+ mice (RLX +/+ M: 27.30±0.32 g; RLX+/+ F: 23.03±0.71 g), but this difference was no longer significant. Themean weight of the RLX +/− mice was between that of the average weightof RLX +/+ and rlx −/− mice. No significant differences were observedbetween the average weight of RLX +/+ and RLX +/− animals, or betweenRLX +/− and rlx −/− mice.

The Effects of Relaxin Gene Knockout on the Size of the MaleReproductive Tract

No significant difference was observed in the weight or size of the malereproductive tract at 1 week of age, which was represented primarily bythe testis (Table 1). By 1 month of age the collected male genital tractwas composed of the testis, epididymis, prostate, seminal vesicle,ductus deference and attached fat. Although no difference in the overallweight of the reproductive tract was observed at one month of age,differences in size of the testis and prostate, derived from 1 monthnull mutant mice, were approximately 20% and 30% smaller, respectively,than the corresponding tissues of RLX wildtype animals, while the sizeof the epididymis, derived from rlx −/− mice was significantly (p<0.05)smaller than that obtained from RLX +/+ animals. No differences werenoted, however, in the size of the seminal vesicle between tissuesamples collected from RLX +/+ and rlx −/− mice.

By the time the mice had reached adulthood (3 months of age), asignificant (p<0.05) difference in the overall weight of the malereproductive tract as well as in the size of individual organs wasobserved (Table 1). The weight of the reproductive tract in normal miceincreased 248.6% from 1 month to adulthood and represented 3.37% of thetotal body weight. During this time, the weight of the reproductivetract from male RLX null mutant mice only increased by 187.9% andrepresented 2.33% of the total body. This latter finding implied thatthe reproductive tract of male rlx −/− mice represented a 31% decreasein % body weight. The size of the testis, epididymis, prostate andseminal vesicle from rlx −/− mice were all smaller (p<0.05) than theirrespective counterparts, derived from RLX +/+ mice.

TABLE 1 The weight and size of the male reproductive tract from 1 weekof age to adulthood (6.5 months). RLX +/+ % of Body rlx −/− % of Body[Mean ± SE(n)] Weight [Mean ± SE(n)] Weight 1 Week of Age: Weight (g):Overall 0.017 ± 0.001(10) 0.43 0.018 ± 0.002(15) 0.53 Size (Area; mm²):Testis  1.30 ± 0.30 (5)¹ —  1.41 ± 0.23 (5)¹ — 1 Month of Age: Weight(g): Overall  0.37 ± 0.02(11) 1.81  0.33 ± 0.01 (11) 1.77 Size (Area;mm²): Testis 16.36 ± 1.58 (9)¹ — 12.91 ± 0.96 (8)¹ — Epididymis 10.73 ±2.22 (7)² —  4.62 ± 0.64 (7)²* — Prostate  9.20 ± 1.20 (4)³ —  6.30 ±0.54 (4)³ — Seminal Vesicle  1.50 ± 0.25 (4)⁴ —  1.69 ± 0.22 (4)⁴ — 3Months of Age: Weight (g): Overall  0.92 ± 0.06(14) 3.37  0.62 ± 0.03(11)* 2.33 Size (Area; mm²): Testis 27.21 ± 1.52 (12)¹ — 17.31 ± 2.28(10)¹ — Epididymis 23.62 ± 3.19 (11)² — 13.71 ± 2.74(10)²* — Prostate20.89 ± 1.20 (6)³ — 15.03 ± 1.60 (6)³* — Seminal Vesicle 25.75 ± 3.58(7)⁴ —  14.6 ± 2.88 (5)⁴* — The size of each ¹ testis and ³ prostate wasmeasured by its area, multiplying the tissue length by width. The size(area) of the epididymis and seminal vesicle could not be measuredaccurately, so a close approximation of each tissue was calculated asfollows: epididymis (by measuring the length of the tissue by the widthof the epididymis head²); seminal vesicle (by measuring the length ofeach organ by the average width of the tissue⁴). *denotes p < 0.05.

The Effects of Relaxin Gene Knockout on Histology of the MaleReproductive Tract

H&E & Masson Trichrome Staining: Male reproductive tissue sections fromRLX +/+ and rlx −/− mice, were first observed for differences in spermmaturation, tubule size/compactness (testis, epididymis) and collagen.

Testis: At 1 week of age, the seminiferous tubules (the compartmentscontaining germ cells/spermatocytes, Sertoli cells and where spermmaturation occurs) of relaxin wildtype animals were smaller, morecylindrical in shape (Table 2) and mainly supported by a thin layer ofcollagen surrounding the tunica albuginea (the membrane that covers theoval body) of each organ. In comparison, tubules derived from rlx −/−mouse testes were much larger and elongated, but were completelysurrounded and supported by collagen within the testis. This immediatelysuggested a difference in the internal organization of the testis in theabsence of relaxin, even just after birth. No immature sperm though weredetected in either group of tissue sections at one week of age. By onemonth of age, testis tubules derived from RLX +/+ mice were larger insize (compared to 1 week tubule size), were slightly less compact andcontained mainly immature sperm. In comparison, the tubules derived from1 month old RLX homozygous mice were no different to tubule sizesmeasured from 1 week old knockout mice and contained less immature spermcompared to samples derived from RLX +/+ mice. This further suggestedthat relaxin null mutant mice were undergoing a process of delayed spermmaturation which was further confirmed when comparing three month tissuesections; sections derived from RLX +/+ mouse tissues contained largertubules (compared to 1 month tubule size) with mainly mature sperm.Conversely, sections derived from rlx −/− mice contained slightlysmaller tubules (which were indifferent to tubule sizes derived from 1week/1 month rlx −/− mice) with less mature sperm (compared to 3 monthRLX +/+ tissue sections) and still some immature sperm. The level ofsperm maturity in testes derived from RLX +/+ mice was shown to besignificantly (p<0.05) greater than the level of sperm maturationobserved in the testes of rlx −/− mice (see Table 2 for grading scaleused). It was also noted that the level of sperm maturation in adult RLXknockout mouse testes resembled that observed in immature (1 month) RLXwildtype mouse tissues. From 1-3 months of age, the testis of normalmice continued to be surrounded by a thin layer of collagen, coveringthe tunica albuginea; however, in some tissues a scattered thin liningof collagen was also observed to support the seminiferous tubules. Theinternal layering of collagen though, was found to decrease with age. Inrlx −/− mice, the collagen detected in-between tubular structures hadregressed due to the larger tubular sized structures within the testis,but was still more dense and consistent compared to that observed intissue samples derived from wildtype animals. It is postulated that theincreased collagen observed in the reproductive tract of RLX null mutantmice at a very young age may cause tissues to be more rigid and firm,which may be linked to the changes in tubular composition and spermmaturation that are discussed above. Interestingly, the presence ofrelaxin in normal adult mice also induced a loosening (increasedinterstitial spacing) of the seminiferous testis tubular structurescompared to that observed from normal immature (1 month) mice. Incomparison, there was no difference in tubule organization(size/compactness) from testes derived from 1 month and 3 month rlx −/−mice. Additionally, 1 month, and to an extent 3 month, testis tubulesderived from rlx −/− mice appeared to contain an increased number ofdead cells, compared to tubular cells derived from RLX wildtype animals.The increased number of dead cells seen in immature tissues werevariable between animals but were consistently detected as the micematured with age.

Epididymis: No significant differences in tubule size were noted in theepididymis of RLX +/+ and rlx −/− reproductive tracts at 1 month and 3months of age. By adulthood however, tubule compactness was more evidentin tissues derived from RLX homozygous mice as compared to tissuesderived from RLX wildtype animals. The epididymis tubules of rlx −/−mice were also supported to a greater extent by connective tissue.Tubular structures derived from 3 month RLX +/+ mouse tissues were looseand only partially maintained by collagen. Conversely, tissue sectionsobtained from RLX knockout animals were shown to have more compacttubules that were fairly well enclosed by thin layers of collagen. As inthe case of the testis, tissues obtained from rlx −/− mice were upheldby an increased concentration of collagen, at all ages observed,compared to tissue samples derived from RLX +/+ mice. As shown in Table2, it was also noted that the density of mature sperm (in epididymistubules) was less in tissue sections derived from RLX null mutant miceat 1 month and 3 months of age, compared to that obtained from RLXwildtype mice, at each respective age group. This difference, however,was statistically insignificant. This decrease in mature sperm in theepididymis of mice lacking relaxin was most likely attributed to thedelayed sperm maturation process that took place in the testis of theseanimals (Table 2).

TABLE 2 The effects of relaxin gene knockout on tissue compartment andsperm maturation, during murine growth and development Age RLX +/+ rlx−/− Tissue [months] [Mean ± SE (n)] [Mean ± SE (n)] Testis: Tubulesize^(a) [0.23] 2.20 ± 0.32 (5) 2.60 ± 0.20 (5) [1] 2.92 ± 0.17 (9) 2.70± 0.13 (8) [3] 3.64 ± 0.08 (9) 2.86 ± 0.18 (10)* [6.5] 3.17 ± 0.17 (3)3.08 ± 0.08 (3) Tubule compactness^(b) [0.23] 3.90 ± 0.10 (5) 3.45 ±0.20 (5) [1] 3.56 ± 0.15 (9) 2.66 ± 0.32 (8) [3] 1.75 ± 0.28 (9) 2.60 ±0.17 (8)* [6.5] 2.33 ± 0.08 (3) 3.66 ± 0.09 (4)* Sperm maturation^(c)[0.23] 0 (5) 0 (5) [1] 1.78 ± 0.36 (9) 0.94 ± 0.20 (8) [3] 3.78 ± 0.15(9) 2.20 ± 0.29 (10)* [6.5] 3.75 (3) 3.56 ± 0.12 (4) Epididymis: Tubulesize^(a) [1] 2.48 ± 0.24 (7) 2.74 ± 0.36 (7) [3] 2.47 ± 0.22 (8) 2.92 ±0.23 (7) [6.5] 2.67 ± 0.17 (3) 2.88 ± 0.13 Tubule compactness^(b) [1]3.16 ± 0.28 (7) 3.01 ± 0.25 (7) [3] 2.68 ± 0.28 (8) 3.24 ± 0.28 (7)[6.5] 2.33 ± 0.44 (3) 3.00 ± 0.18 (4) Sperm maturation^(c) [1] 0.94 ±0.06 (8) 0.75 ± 0.10 (8) [3] 3.56 ± 0.13 (9) 3.05 ± 0.17 (10) [6.5] 3.83± 0.08 (3) 3.63 ± 0.07 (4) Table 2: Tissue sections were stained with H& E and graded as follows: ^(a)Tubule size - 1 = all tubules small andcircular in shape; 2 = most tubules slightly elongated compared to 1 butsome smaller circular tubules are observed; 3 = tubules grosslyelongated compared to 1, but some smaller circular and mid-sized tubulesare observed; 4 = all tubules grossly elongated compared to 1 and 2.^(b)Tubule compactness - 1 = all tubules loose; 2 = proportion of loosetubules greater than proportion of compact tubules; 3 = proportion ofcompact tubules greater than proportion of loose tubules; 4 = alltubules compact. ^(c)Sperm maturation - 0 = no sperm detected; 1 = onlyimmature sperm detected; 2 = proportion of immature sperm greater thanproportion of mature sperm; 3 = proportion of mature sperm greater thanproportion of immature sperm; 4 = only mature sperm detected. Thenumbers presented are the mean ± SE (n) of the grading scale used foreach parameter measured. *denotes p < 0.05.

TABLE 3 The effects of relaxin gene knockout on collagen maturation,during murine growth and development Age RLX +/+ rlx −/− Tissue [months][Mean ± SE (n)] [Mean ± SE (n)] Testis^(a): [0.23] 1 (4) 2.00 ± 27 (4)[1] 1.09 ± 0.07 (8) 1.61 ± 0.25 (7) [3] 1.06 ± 0.06 (9) 1.28 ± 0.09 (8)[6.5] 1.75 ± 0.14 (3) 2.31 ± 0.12 (4) Epididymis^(a): [1] 2.41 ± 0.33(7) 2.57 ± 0.39 (7) [3] 1.82 ± 0.21 (8) 2.86 ± 0.29 (8)* [6.5] 2.92 ±0.08 (3) 3.06 ± 0.19 (4) Prostate^(a): [1] 1 (3) 2.33 ± 0.17 (3) [3]1.25 ± 0.17 (6) 2.25 ± 0.28 (6) [6.5] 1.5 (3) 2.69 ± 0.37 (4)* SeminalVesicle^(a): [1] 1 (3) 1.50 ± 0.25 (4) [3] 1.14 ± 0.09 (7) 1.70 ± 0.19(5) Table 3: Tissue sections were stained with Masson trichrome stainand graded as follows: ^(a)Collagen - 1 = only thin lining of collagensurrounding the outer layer of structures; 2 = additional lining ofcollagen surrounding internal components of tissue; 3 = thicker liningof collagen surrounding outer layer and internal components of tissues;4 = 3 + spacing between tubules/components filled with collagen. Thenumbers presented are the mean ± SE (n) of the grading scale. *denotes p< 0.05

Prostate: At 1 month of age, no significant differences were noted inthe structure of the prostate between RLX +/+ and rlx −/− derivedtissues. However, the spacing between the ducts of prostate glands fromnormal animals was associated with only trace amounts of collagen. Incontrast, the ducts of prostate glands from rlx −/− mice were intervenedby layers of loose connective tissue. By adulthood (3 months), rlx −/−mice contained smaller prostates with smaller glands and ducts. Theducts of tissues derived from RLX knockout mice were more compact andcompletely supported by connective tissue, while the ducts obtained fromRLX wildtype mice were more spread out (loose) and still supported bylittle collagen. Furthermore, the ducts of the adult prostate obtainedfrom rlx −/− mice appeared to have smaller epithelium, containing lesscells, while the ducts of RLX +/+ prostate samples contained largerepithelial layers/glandular tissue. These results added to our initialfindings on the testis and epididymis in implying that a delayedmaturation process of male reproductive tissues was taking place in micelacking a functionally active relaxin gene and involved an accumulationof collagen within these tissues.

Detection of Cell Apoptosis by Antibody Staining:

Based on observations made from H&E staining, whereby tissues derivedfrom rlx −/− mice underwent decreased sperm maturation and increasedcell death (testis) and contained decreased epithelial (cell) layers(prostate), it was decided to investigate whether the increased celldeath observed were the result of apoptotic pathways.

Bax Antibody Staining: Overexpression of Bax accelerates apoptotic deathinduced by cytokine deprivation and also counters the death repressoractivity of Bcl-2 (Krajewski et al., Am. J. Pathol. 145:1323-36 (1994)).Using a Bax monoclonal antibody, the in vivo distribution of the Baxprotein was evaluated in the male reproductive tract. Testis:Seminiferous tubules derived from immature (1 month) wildtype animalsshowed weak immunostaining for Bax (dark brown cell staining), which wasprimarily observed in the germinal cells near the basement membrane.These findings are consistent with those of previous reports (Ben-Hur etal., Calcif. Tiss. Int. 53:91-96 (1996)). In comparison, an increasednumber of cells were stained positive for Bax in testes derived from rlx−/− mice. The number of Bax positive cells were counted using ahemacytometer and shown to be significantly greater in the testis of rlx−/− mice (32.7±4.8 cells/testis (n=6)) compared to the number ofapoptotic cells observed from RLX +/+ mouse-derived tissues (13.1±3.5cells/testis (n=6)). While increased Bax staining was consistentlyobserved in rlx −/− tissues, the number of tubules stained positive forBax varied between tissue samples, and some tubules were also observedto be Bax-free. As the male reproductive tract matured, adult RLXwildtype mice testes showed decreased staining for Bax (3.4±1.4cells/testis (n=6)) which correlated with the increased level of spermand tissue maturity that these animals underwent. Conversely, the slowerrate of tissue maturity observed in the rlx −/− mouse reproductive tractcorrelated with a further increase in Bax staining in 3 month RLXknockout mice (44.4±8.1 cells/testis (n=6)). Many of the observed cellsappeared to be at the final stages of apoptosis, perhaps representingapoptotic bodies. Bax staining was not associated with mature spermcells though. Epididymis: A more intense Bax staining was present in theintracellular membrane of epithelial cells of epididymis tubules derivedfrom 1 month RLX +/+ mouse tissues. The same tubules derived from 1month rlx −/− mouse tissues contained a relatively stronger level of Baxstaining, which was further maintained, if not increased in 3 monthtissue sections. In contrast, the epididymis of adult wildtype micecontained a weaker expression of Bax staining, compared to tissuesderived from 1 month normal mice, as the tissue matured with age.Prostate: The epithelial cells of the prostate of normal 1 month and 3month animals showed weak positive staining for Bax. As with the othertissues studied, cells specifically within the epithelial layer (ofprostate ducts) of rlx −/− animals showed increased staining for Bax inboth immature (1 month) and adult (3 month) tissues. These findingssuggested that relaxin may have played a novel role in the regulation ofcell apoptosis within the male reproductive tract, however, further workusing a separate antibody to detect cell apoptosis was conducted toconfirm the actions of relaxin.

Caspase-9, a central death protease, belongs to a unique family ofcysteine proteases that differ in sequence, structure and substratespecificity to other described protease families. The caspase familymembers (which are usually involved in a cascade of proteolytic cleavageevents) function as key components of the apoptotic machinery by actingto destroy specific target proteins which are critical to cellularactivity. Testis:

Moderate caspase-9 staining was observed in 1 month RLX +/+ testes(28.1±5.6 cells/testis (n=7)), which appeared to be primarily associatedwith the germinal cells within the seminiferous tubules, rather thanwith spermatagonia or spermatocytes. As with Bax, a significantly(p<0.05) increased level of caspase-9 staining was detected in 1 monthrlx −/− mouse testes (76.6±10.2 cells/testis (n=6)), although heavycaspase-9 staining was associated with few tubules, while many tubuleswere observed to contain little or no caspase-9 protein. With age, thelevel of caspase-9 detected in normal immature tissues was consistentlyfound in older tissues at 3 months of age (26.9±4 cells/testis (n=6)).However, as the testis increased in size, the number of apoptotic cellsdetected represented a smaller fraction of the tissue with age. Testistubules derived from rlx −/− mice contained slightly fewer apoptoticcells at 3 months (61.6±9.8 cells/testis (n=5)), compared to the densityof caspase-9 stained cells in 1 months tissues. The number of positivelystained apoptotic cells from RLX knockout mouse tissues though, remainedsignificantly (p<0.05) greater than their wildtype counterparts at allages studied. Epididymis: No staining for caspase-9 was detected from 1month RLX +/+ epididymis tubules, while trace amounts of positive cellswere observed in rlx −/− tissue sections. However, the positivelystained cells were sparsely scattered and were detected in theepithelial layer of the tubules. With age, clear staining for caspase-9was not detected in 3 month RLX +/+ and rlx −/− mouse tissues. Prostate:No staining for caspase-9 was identified in the prostate of RLX +/+ andrlx −/− tissues at all ages (1-3 months) investigated. Nevertheless, theobtained results suggested for the first time, that relaxin is linked tothe regulation of cell apoptosis. Additional work though was required toestablish whether relaxin was involved in cell proliferation pathways.

Detection of Cell Proliferation by Antibody Staining:

PCNA staining: The proliferating cell nuclear antigen (PCNA) antibodywas used to detect cell proliferation in the male reproductive tractbased on its ability to associate with nuclear regions where DNAsynthesis is occurring. Testis: PCNA was detected in immature and matureseminiferous tubules, although no significant differences in PCNAstaining were detected in testis tubules derived from RLX +/+ and rlx−/− mice at 1 month and 3 months of age. In immature tissues, PCNAstaining is usually highly expressed in mitotically active spermatogoniaand occasionally in some Sertoli cells, which corresponds toproliferative activity. In this study, these cells were more uniformlyand continuously labeled for PCNA even well into murine adulthood, whichperhaps reflects the ability of both groups to be able to initiatereproduction, via the constant activation of these proliferative cells.Epididymis: No staining for PCNA was detected in the epididymis derivedfrom either RLX +/+ or rlx −/− mice at 1 month and 3 months of age.These findings are consistent with the role of the epididymis in actingas a reservoir for mature sperm. Prostate: Weak staining for PCNA wasassociated with the epithelial cells of prostate ducts derived from 1month RLX +/+ and rlx −/− mice. However, no staining for PCNA wasdetected from either group at 3 months of age. While cell proliferationstudies were limited to some reproductive tissues, the accumulatedfindings perhaps confirmed that relaxin was most likely to play aninfluential role in the regulation of cell apoptosis, rather than oncell proliferation.

The Effects of Relaxin Gene Knockout on Histology of Other Body Tissues

RLX wildtype (RLX +/+) and RLX gene knockout (rlx −/−) male and femalemice were generated from RLX heterozygous (RLX +/−) parents, asdescribed above. Mice were weighed and sacrificed at 1 week, 1 month and3 months of age for tissue collection. The following tissues, includingthe brain, heart, liver, kidneys, thymus, spleen and male reproductivetract were collected, weighed and placed into 10% formalin for detailedhistological analysis. A summary of the weights of these tissues areshown in Tables 4-8.

At 1 week of age, male rlx −/− mice contained significantly (p<0.05)smaller livers, kidneys and spleens compared to RLX +/+ animals, butthese weight differences were no longer apparent at 1 month of age.Instead, the thymus of 1 month old rlx −/− male mice was smaller(p<0.05) than their RLX +/+ counterparts. This weight difference was nolonger apparent at 3 months of age. In the case of the male reproductivetract, no significant differences were noted at 1 week or 1 month of agebetween RLX +/+ and rlx −/− groups, however by 3 months of age, thereproductive tract of rlx −/− mice was significantly (p<0.05) smallerthan that obtained from RLX +/+ animals.

No notable differences were observed between 1 week old RLX +/+ and rlx−/− female mice, however, by 1 month of age, rlx −/− female mice hadsignificantly smaller livers than their RLX +/+ counterparts. By threemonths of age, this weight difference was no longer observed and noother significant differences of the other organs were noted.

To determine if the organ and tissue differences in RLX−/− mice (derivedfrom RLX +/− parents) would be accentuated further if rlx −/− offspringwere obtained from RLX−/− parents, RLX +/+ and rlx −/− mice ere obtainedfrom the corresponding set of parents and were sacrificed at 1 week, 1month and 3 months of age. The brain, heart, liver, kidneys, thymus,spleen, male reproductive tract, intestine and lung were collected andweighed. The weights of these organs are shown in Tables 4-8.

It was further noted that, although the weights of organ or tissueappeared to normalize as the mice become older, normal weight alone doesnot equate with a normal organ or tissue. The data show that adiscrepancy in organ (tissue) weight to body weight ratio at any time inthe growth cycle indicates a change in underlying organ or tissuedevelopment or cellular architecture. As one example, a small liver thatis infiltrated (altered) by fibrosis will weigh the same or more than alarger normal liver or one filled with fat. Thus, tissues from brain,heart, liver, kidneys, thymus, spleen, intestine and lung of male andfemale RLX−/− mice (developed in the absence of relaxin) show evidenceof increased apoptosis and extracellular matrix (collagen) accumulation.

TABLE 4 Tissue Comparison of 1 week old (from RLX +/+ & rlx −/− parents,respectively), Represented as Mean ± SE (n): % % body body RLX +/+ Malewt rlx −/− Male wt Body wt (g)  3.85 ± 0.14 (10) —  3.32 ± 0.14 (15) —Rep. Organ (g) 0.017 ± 0.001 (10) 0.43 0.018 ± 0.001 (15) 0.53 Brain (g) 0.24 ± 0.01 (10) 6.31  0.21 ± 0.01 (15) 6.40 Heart (g) 0.023 ± 0.002(10) 0.58 0.020 ± 0.001 (15) 0.60 Liver (g)  0.12 ± 0.01 (10) 3.13 0.094± 0.01 (15)* 2.82 L. Kidney (g) 0.023 ± 0.001 (10) 0.59 0.018 ± 0.001(15)* 0.54 R. Kidney (g) 0.023 ± 0.001 (10) 0.59 0.019 ± 0.001(15)* 0.56Spleen (g) 0.033 ± 0.002 (10) 0.59 0.018 ± 0.001 (15)* 0.54 Thymus (g)0.020 ± 0.001 (10) 0.51 0.018 ± 0.001 (15) 0.54 % % body body RLX +/+Female wt rlx −/− Female wt Body wt (g)  3.45 ± 0.19 (9) —  3.51 ± 0.15(9) — Brain (g)  0.22 ± 0.01 (9) 6.23  0.22 ± 0.01 (9) 6.11 Heart (g)0.023 ± 0.002 (9) 0.67  0.02 ± 0.001 (9) 0.57 Liver (g) 0.085 ± 0.01 (9)2.46  0.10 ± 0.01 (9) 2.85 L. Kidney (g) 0.019 ± 0.001 (9) 0.55  0.20 ±0.001 (9) 0.56 R. Kidney (g) 0.020 ± 0.001 (9) 0.57  0.20 ± 0.001 (9)0.57 Spleen (g) 0.020 ± 0.002 (9) 0.57 0.017 ± 0.002 (9) 0.48 Thymus (g)0.017 ± 0.002 (9) 0.48 0.017 ± 0.001 (9) 0.47 *=> p < 0.05

TABLE 5 Tissue Comparison 1 month old mice (from RLX+/− parents),Represented as Mean ± SE (n): % % body body RLX +/+ Male wt rlx −/− Malewt Body wt (g) 20.54 ± 0.82 (10) — 18.29 ± 0.84 (10) — Rep. Organ (g) 0.37 ± 0.02 (11) 1.81  0.33 ± 0.01 (11) 1.78 Brain (g)  0.37 ± 0.01(10) 1.81  0.37 ± 0.01 (10) 2.03 Heart (g)  0.13 ± 0.004 (10) 0.62  0.13± 0.01 (10) 0.69 Liver (g)  1.17 ± 0.05 (10) 5.70  1.10 ± 0.05 (10) 5.99L. Kidney (g)  0.16 ± 0.01 (10) 0.77  0.14 ± 0.01 (10) 0.78 R. Kidney(g)  0.17 ± 0.01 (10) 0.82  0.14 ± 0.01 (10) 0.78 Spleen (g)  0.12 ±0.01 (10) 0.57  0.12 ± 0.01 (10) 0.66 Thymus (g)  0.06 ± 0.01 (10) 0.280.075 ± 0.005 (10) 0.41 % % body body RLX +/+ Female wt rlx −/− Femalewt Body wt (g) 16.93 ± 0.45 (10) — 16.04 ± 0.61 (9) — Brain (g)  0.36 ±0.01 (9) 2.14  0.36 ± 0.01 (9) 2.23 Heart (g)  0.09 ± 0.003 (10) 0.53 0.10 ± 0.003 (9) 0.60 Liver (g)  0.90 ± 0.02 (10) 5.32  0.78 ± 0.02(9)* 4.84 L. Kidney (g)  0.13 ± 0.005 (10) 0.78  0.12 ± 0.003 (9) 0.76R. Kidney (g)  0.13 ± 0.004 (10) 0.78  0.13 ± 0.01 (9) 0.80 Spleen (g) 0.08 ± 0.004 (10) 0.45  0.08 ± 0.01 (9) 0.50 Thymus (g)  0.07 ± 0.01(10) 0.39  0.07 ± 0.01 (9) 0.42 *=> p < 0.05

TABLE 6 Tissue Comparison of 3 month old male mice (from RLX +/+ & rlx−/− parents, respectively), Represented as Mean ± SE (n): % % body bodyRLX +/+ Male wt rlx −/− Male wt Body wt (g) 27.41 ± 0.39 (14) —  26.57 ±0.51 (11) — Rep. Organ (g)  0.92 ± 0.06 (14) 3.37   0.62 ± 0.02 (11)2.33 Brain (g)  0.37 ± 0.01 (10) 1.34   0.37 ± 0.01 (10) 1.40 Heart (g) 0.15 ± 0.003 (10) 0.56   0.14 ± 0.01 (10) 0.54 Liver (g)  1.32 ± 0.08(10) 4.80   1.28 ± 0.04 (10) 4.82 L. Kidney (g)  0.24 ± 0.01 (10) 0.87  0.21 ± 0.01 (10) 0.81 R. Kidney (g)  0.23 ± 0.01 (10) 0.83 0.0.22 ±0.01 (10) 0.82 Spleen (g)  0.08 ± 0.01 (10) 0.29   0.08 ± 0.003 (10)0.29 Thymus (g) 0.056 ± 0.01(10) 0.20  0.051 ± 0.003 (10) 0.19 % % bodybody RLX +/+ Female wt rlx −/− Female wt Body wt (g) 23.14 ± 1.08 (10) — 21.62 ± 0.39 (9) — Brain (g)  0.38 ± 0.01 (10) 1.64   0.39 ± 0.01 (9)1.80 Heart (g)  0.10 ± 0.004 (10) 0.44   0.11 ± 0.004 (9) 0.49 Liver (g) 1.07 ± 0.05 (10) 4.61   1.01 ± 0.004 (9) 4.66 L. Kidney (g)  0.16 ±0.004 (10) 0.67   0.15 ± 0.006 (9) 0.69 R. Kidney (g)  0.16 ± 0.005 (10)0.70   0.15 ± 0.003 (9) 0.70 Spleen (g)  0.09 ± 0.01 (10) 0.37   0.09 ±0.01 (9) 0.41 Thymus (g)  0.06 ± 0.003 (10) 0.26   0.06 ± 0.003 (9) 0.29*=> p < 0.05

TABLE 7 Tissue Comparison of 1 month old mice (from RLX+/+ and RLX−/−parents, respectively), represented as Mean ± SE (n): % %  body body RLX+/+ Male wt rlx −/− Male wt Body wt (g) 19.92 ± 0.51 (11) — 14.90 ± 1.02(4)* — Rep. Organ (g)  0.48 ± 0.02 (11) 2.42  0.27 ± 0.04 (4) 1.78 Brain(g)  0.41 ± 0.01 (11) 2.06  0.40 ± 0.01 (4) 2.70 Heart (g)  0.12 ± 0.004(11) 0.61  0.12 ± 0.01 (4) 0.81 Liver (g)  1.11 ± 0.04 (11) 5.58  0.72 ±0.06 (4)* 4.85 L. Kidney (g)  0.14 ± 0.01 (11) 0.68  0.10 ± 0.01 (4)0.65 R. Kidney (g)  0.13 ± 0.01 (10) 0.67  0.10 ± 0.01 (4) 0.65 Spleen(g)  0.11 ± 0.01 (11) 0.53  0.11 ± 0.02 (4) 0.72 Thymus (g)  0.08 ± 0.01(11) 0.42 0.085 ± 0.003 (4) 0.57 Gut (g)  0.48 ± 0.03 (11) 2.40  0.56 ±0.07 (4) 3.78 Lung (g)  0.19 ± 0.01 (11) 0.97  0.16 ± 0.01 (4) 1.06 % %body body RLX +/+ Female wt rlx −/− Female wt Body wt (g) 16.68 ± 0.38(11) — 13.15 ± 1.40 (5)* — Brain (g)  0.41 ± 0.01 (11) 2.48  0.40 ± 0.02(5) 3.07 Heart (g)  0.10 ± 0.003 (11) 0.59  0.10 ± 0.003 (5) 0.76 Liver(g)  0.90 ± 0.03 (11) 5.39  0.57 ± 0.07 (5)* 4.32 L. Kidney (g)  0.10 ±0.004 (11) 0.61  0.10 ± 0.003 (5) 0.73 R. Kidney (g)  0.10 ± 0.003 (11)0.62  0.10 ± 0.01 (5) 0.75 Spleen (g)  0.10 ± 0.01 (11) 0.57  0.09 ±0.01 (5) 0.68 Thymus (g)  0.10 ± 0.010 (11) 0.59  0.08 ± 0.003 (5) 0.61Gut (g)  0.50 ± 0.04 (11) 2.97  0.51 ± 0.03 (5) 3.86 Lung (g)  0.20 ±0.01 (6) 1.18  0.12 ± 0.01 (4) 0.93 *=> p < 0.05

TABLE 8 Tissue Comparison of 3 month old mice (from RLX+/+ and RLX−/−parents, respectively), represented as Mean ± SE (n): % % body body RLX+/+ Male wt rlx −/− Male wt Body wt (g) 26.10 ± 0.51 (12) — 24.37 (1) —Rep. Organ (g)  0.90 ± 0.03 (12) 3.45  0.82 (1) Brain (g)  0.41 ± 0.01(12) 1.56  0.40 (1) Heart (g)  0.16 ± 0.003 (12) 0.60  0.14 (1) Liver(g)  1.25 ± 0.04 (8) 4.79  1.24 (1) L. Kidney (g)  0.20 ± 0.005 (12)0.75  0.17 (1) R. Kidney (g)  0.20 ± 0.004 (12) 0.75  0.18 (1) Spleen(g) 0.085 ± 0.003 (12) 0.33  0.08 (1) Thymus (g) 0.058 ± 0.004 (12) 0.220.011 (1) Gut (g)  0.62 ± 0.05 (12) 2.36  0.5 (1) Lung (g)  0.25 ± 0.02(7) 0.95  0.2 (1) % % body body RLX +/+ Female wt rlx −/− Female wt Bodywt (g) 22.30 ± 1.63 (12) — 23.73 ± 0.45 (5) — Brain (g)  0.41 ± 0.01(12) 1.86  0.44 ± 0.01 (5) 1.86 Heart (g)  0.13 ± 0.003 (12) 0.59  0.14± 0.002 (5) 0.61 Liver (g)  1.15 ± 0.05 (10) 5.16  1.11 ± 0.003 (5) 4.67L. Kidney (g)  0.14 ± 0.004 (12) 0.62  0.14 ± 0.003 (5) 0.60 R. Kidney(g)  0.16 ± 0.005 (12) 0.62  0.14 ± 0.01 (5) 0.61 Spleen (g)  0.09 ±0.004 (12) 0.40  0.10 ± 0.01 (5) 0.41 Thymus (g)  0.07 ± 0.003 (12) 0.29 0.08 ± 0.010 (5) 0.33 Gut (g)  0.55 ± 0.04 (12) 2.45  0.68 ± 0.04 (5)2.86 Lung (g)  0.21 ± 0.01 (6) 0.96  0.21 ± 0.005 (5) 0.86 *=> p < 0.05

The Effects of Relaxin Gene Knockout on the Histology of Skin

The role of relaxin in the regulation of tissue remodeling was examinedby studying the skin histology in rlx-null mice (rlx−/−). These micewere the progeny of rlx−/− male and female parents. Sequential skinsamples from the ear and dorsum of the back of at least 5 male and 5female mice were stained with H&E and Masson's trichrome stain andexamined at each time point. Similar samples at the same time pointswere obtained from Rlx+/+ mice of the same strain. Upon histologicalexamination of rlx-null mice, the dermis was found to have thickenedprogressively with time and had increased fibrosis throughout thedermis. Dermal samples of rlx-null mice were normal at one week of age;however, by one month early dermal fibrosis was evident. By 3 months ofage there was a marked increase in dermal fibrosis that increased indensity by 6 months of age. These dermal findings were similar in maleand female rlx-null mice.

In these mice, the epidermis was normal, and hair was not altered,except for an initial lighter coat color in rlx-null mice that becameindistinguishable from Rlx +/+ mice by one month of age. Examination ofserum chemistries, hematological parameters and urine from the rlx-nullmice were unremarkable.

These findings support previous observations that relaxin influencesmatrix turnover in vitro and in vivo by altering key matrix molecules,matrix-degrading enzymes and growth factors. Many stromal cells thatproduce interstitial collagens have relaxin receptors, whether derivedfrom male or female sources. Antibody-based assays can detect relaxin inserum at the low picogram level in females during the luteal phase ofthe menstrual cycle, and these levels rise during pregnancy, a time whentissue remodeling is most evident. In contrast, serum relaxin isreported to be undetectable in males.

Rlx-null mice offer the first direct evidence that links relaxin to ageneralized alteration of collagen turnover in normal skin. Theseresults indicate that relaxin may be produced and circulate atbiologically relevant concentrations below current levels of detectionin males and females. Relaxin participates in the ordered maintenance ofmatrix turnover in concert with other matrix-regulating molecules.Relaxin can influence tissue remodeling, promotion of blood vesselformation and stimulation of vasodilatation. These results also indicatethat the relaxin synthetic pathway, and/or relaxin receptor, isassociated with fibrotic conditions, such as scleroderma.

The previous examples are provided to illustrate but not to limit thescope of the claimed inventions. Other variants of the inventions willbe readily apparent to those of ordinary skill in the art andencompassed by the appended claims. All publications, patents, patentapplications and other references cited herein are hereby incorporatedby reference.

1. A method of modulating apoptosis, comprising: administering to asubject in need thereof an effective amount of a relaxin agonist for aperiod of time sufficient to decrease apoptosis in cells expressing arelaxin receptor.
 2. The method of claim 1, wherein the cells are fromthe liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung,the male reproductive tract, or the female reproductive tract.
 3. Themethod of claim 2, wherein the male reproductive tract tissue isprostate, epididymis, seminal vesicles or testes.
 4. The method of claim2, wherein the female reproductive tract tissue is uterus, cervix, theinterpubic ligament, or connective tissues within the pelvic girdle. 5.(canceled)
 6. A method for stimulating maturation, development ordifferentiation of an immature tissue, comprising administering to asubject in need thereof an effective amount of a relaxin agonist for atime period sufficient to stimulate tissue maturation.
 7. (canceled) 8.The method of claim 6, wherein the tissue is male reproductive tracttissue.
 9. The method of claim 8, wherein the tissue is prostatictissue, epididymal tissue, seminiferous tissue, testicular tissue orsperm.
 10. (canceled)
 11. The method of claim 9, wherein the maturationis an increase in cell number in an under-developed testes.
 12. Themethod of claim 9, wherein the number of viable sperm cells increases.13. The method of claim 1, wherein the subject has a relaxin-deficientcondition.
 14. The method of claim 13, wherein the relaxin-deficientcondition is immature tissue, or low sperm count.
 15. The method ofclaim 14, wherein the immature tissue is immature male reproductivetract tissue.
 16. The method of claim 15, wherein the immature malereproductive tract tissue is underdeveloped testes.
 17. (canceled) 18.The method of claim 1, wherein the relaxin agonist is relaxin, a relaxinanalog, a small molecule relaxin effector, or a relaxin nucleic acid.19. The method of claim 18, wherein the relaxin is vertebrate relaxin orhuman relaxin.
 20. (canceled)
 21. The method of claim 1, wherein theadministering is by infusion, injection, oral delivery, nasal delivery,intrapulmonary delivery, rectal delivery, transdermal delivery,interstitial delivery or subcutaneous delivery.
 22. The method of claim21, wherein the administering is by delayed release delivery. 23-24.(canceled)
 25. The method of claim 1, wherein the administeringcomprises delivery of a vector encoding the relaxin agonist.
 26. Themethod of claim 1, wherein the vector is an expression vector, andwhereby the relaxin agonist is expressed in the cells.
 27. (canceled)28. The method of claim 1, wherein the subject is a post-pubescentsubject or a pre-pubescent subject. 29-52. (canceled)